Method and substances for isolating mirnas

ABSTRACT

A capture probe suitable for use with a method for isolating miRNAs. A method for isolating an miRNA of interest from a sample comprising the miRNA of interest comprising providing the capture probe. A method for identifying an miRNA of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication 60/709,861, titled “Method and Substances for the Isolation,Amplification and Detection of miRNAs,” filed Aug. 19, 2005, thecontents of which are incorporated in this disclosure by reference intheir entirety.

BACKGROUND

MicroRNAs (miRNAs) are small, generally between 18 and 24 residues,polyribonucleotides derived from longer hairpin noncoding transcripts ineukaryotes. miRNAs play a significant role in cellular developmental anddifferentiation pathways. Consequently, there have been considerableefforts made to understand and characterize the temporal, spatial andcellular expression levels and patterns of expression of miRNAs toascertain their precise role in cellular development and differentiationin both normal and disease states.

miRNAs are currently studied by, first, obtaining the total RNA from asample. Next, the total RNA is fractionated into subpopulations by gelelectrophoresis or by chromatographic fractionation and size selectiveelution. Then, the appropriate section of the gel is cut, and the 18-24RNAs are eluted from the gel, or the eluted fraction containing singlestranded RNAs in the size range of 18-24 ribonucleotides is collected.Next, the RNAs are isolated by precipitation and the miRNAs arecharacterized.

Disadvantageously, however, these methods do not work well when theamount of sample is small, such as samples from tumor tissue or biopsymaterial. Further, characterization of the miRNAs isolated by presentmethods usually comprises a several step amplification procedurefollowed by detection, quantitation, cloning and sequencing. Because ofthe large number of steps in these processes and the notoriousinefficiencies associated with the repeated purification, the isolationand identification of miRNAs using present methods is time consuming,relatively expensive, requires relatively large amounts of material andis not fully representative of the population of miRNAs expressed withina small sample, such as within a biopsy of a tumor. Additionally, thepresent methods are not specific to isolating and identifying an miRNA,and therefore, often isolate and identify siRNA, tRNA, 5S/5.8SrRNA anddegraded RNA from additional cellular RNAs.

Therefore, there is the need for an improved method for isolation andidentification of miRNAs that is not associated with thesedisadvantages.

SUMMARY

According to one embodiment of the present invention, there is provideda capture probe suitable for use with a method for isolating miRNAs. Thecapture probe comprises: a) a first adapter segment having a firstadapter segment sequence, the first adapter segment comprising a 3′ endand a 5′ end; b) a second adapter segment having a second adaptersegment sequence, the second adapter segment comprising a 3′ end and a5′ end; and c) an miRNA binding segment having an miRNA binding segmentsequence, where the miRNA binding segment is substantially complementaryto, and capable of hybridizing to, one or more than one miRNA ofinterest by Watson-Crick base pairing, where the 5′ end of the firstadapter segment is connected to the 3′ end of the miRNA binding segment,and where the 3′ end of the second adapter segment is connected to the5′ end of the miRNA binding segment.

In one embodiment, the capture probe comprises a substance selected fromthe group consisting of one or more than one type of polynucleotide, oneor more than one type of polynucleotide analog, and a combination of oneor more than one type of polynucleotide and polynucleotide analog.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes ofthe set of capture probes is a capture probe according to the presentinvention, where each of the capture probes comprises identical-firstadapter segment sequences, where each of the capture probes of the setof capture probes comprises identical miRNA binding segment sequences,and where each of the capture probes of the set of capture probescomprises identical second adapter segment sequences.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, and where the setcomprises at least one capture probe comprising an miRNA binding segmentthat is substantially complementary to, and capable of hybridizing to,each miRNA from a single public database.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical firstadapter segment sequences, where the first capture probe and the secondcapture probe have identical miRNA binding segment sequences, and wherethe first capture probe has a second adapter segment sequence that isdifferent from the second adapter segment sequence of the second captureprobe.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical firstadapter segment sequences, where the first capture probe and the secondcapture probe have identical second adapter segment sequences, and wherethe first capture probe has an miRNA binding segment sequence that isdifferent from the miRNA binding segment sequence of the second captureprobe.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical miRNAbinding segment sequences, where the first capture probe and the secondcapture probe have identical second adapter segment sequences, and wherethe first capture probe has a first adapter segment sequence that isdifferent from the first adapter segment sequence of the second captureprobe.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical firstadapter segment sequences, where the first capture probe has an miRNAbinding segment sequence that is different from the miRNA bindingsegment sequence of the second capture probe, and where the firstcapture probe has a second adapter segment sequence that is differentfrom the second adapter segment sequence of the second capture probe.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical miRNAbinding segment sequences, where the first capture probe has a firstadapter segment sequence that is different from the first adaptersegment sequence of the second capture probe, and where the firstcapture probe has a second adapter segment sequence that is differentfrom the second adapter segment sequence of the second capture probe.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical secondadapter segment sequences, where the first capture probe has a firstadapter segment sequence that is different from the first adaptersegment sequence of the second capture probe, and where the firstcapture probe has a miRNA binding segment sequence that is differentfrom the miRNA binding segment sequence of the second capture probe.

According to another embodiment of the present invention, there isprovided a set of capture probes, where each of the capture probes is acapture probe according to the present invention, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe has a first adapter segment sequence that isdifferent from the first adapter segment sequence of the second captureprobe, where the first capture probe has an miRNA binding segmentsequence that is different from the miRNA binding segment sequence ofthe second capture probe, and where the first capture probe has a secondadapter segment sequence that is different from the second adaptersegment sequence of the second capture probe.

According to another embodiment of the present invention, there isprovided a capture probe according to the present invention, where thefirst adapter segment, or the second adapter segment, or both the firstadapter segment and the second adapter segment are between 6 and 16residues.

According to another embodiment of the present invention, there isprovided a capture probe according to the present invention, where thefirst adapter segment, or the second adapter segment, or both the firstadapter segment and the second adapter segment further comprise asequence that is a polynucleotide synthesis promoter motif for apolynucleotide polymerase, or that is complementary to a polynucleotidesynthesis promoter motif for a polynucleotide polymerase. In oneembodiment, the polynucleotide synthesis promoter motif is a motif for apolynucleotide synthesis promoter selected from the group consisting ofT7, SP6, a T3 DNA dependent RNA polymerase, a type 2 RNA polymerase ofE. coli and single stranded DNA dependent N4 RNA polymerase.

According to another embodiment of the present invention, there isprovided a capture probe according to the present invention, where thefirst adapter segment, or the second adapter segment, or both the firstadapter segment and the second adapter segment further comprise arestriction site motif. In one embodiment, the restriction site motif isacted upon by a restriction enzyme selected from the group consisting ofNot I, Xho I, Xma I and Nhe I.

According to another embodiment of the present invention, there isprovided a capture probe according to the present invention, where thefirst adapter segment, or the second adapter segment, or both the firstadapter segment and the second adapter segment further comprise a solidphase binding group to immobilize the capture probe to a solid phase. Inone embodiment, the solid phase binding group is at or near the 3′ endof the first adapter segment. In another embodiment, the solid phasebinding group is at or near the 5′ end of the second adapter segment. Inanother embodiment, the solid phase binding group immobilizes thecapture probe to the solid phase covalently. In another embodiment, thesolid phase binding group immobilizes the capture probe to the solidphase non-covalently. In another embodiment, the solid phase bindinggroup comprises biotin or an analog of biotin.

According to another embodiment of the present invention, there isprovided a capture probe according to the present invention, where themiRNA binding segment consists of 18 or 19 or 20 or 21 or 22 or 23 or 24residues selected from the group consisting of DNA, RNA, chimericDNA/RNA, DNA analogs and RNA analogs. In another embodiment, the miRNAof interest that the miRNA binding segment is substantiallycomplementary to, and capable of hybridizing to, is selected from apublic database. In another embodiment, the miRNA of interest is aeucaryotic miRNA. In another embodiment, the miRNA of interest is aprimate miRNA. In another embodiment, the miRNA of interest is a humanmiRNA. In another embodiment, the miRNA binding segment is exactly thecomplement to the miRNA of interest in both length and sequence. Inanother embodiment, the miRNA binding segment is more than 90%complementary to a segment of the miRNA of interest of the same lengthas the miRNA of interest sequence. In another embodiment, the miRNAbinding segment is more than 80% complementary to a segment of the miRNAof interest of the same length as the miRNA of interest sequence. Inanother embodiment, the first adapter segment has a first adaptersegment sequence according to SEQ ID NO:1. In another embodiment, thesecond adapter segment has a second adapter segment sequence accordingto SEQ ID NO:2.

According to another embodiment of the present invention, there isprovided a method for isolating an miRNA of interest from a samplecomprising the miRNA of interest. The method comprises: a) providing asample comprising the miRNA of interest; b) providing a capture probeaccording to the present invention; c) providing a first linker and asecond linker; d) combining the sample, the capture probe, the firstlinker and the second linker; e) allowing the first linker to hybridizewith the first adapter segment, the miRNA of interest to hybridize withthe miRNA binding segment, and the second linker to hybridize with thesecond adapter segment; f) ligating the 3′ end of the first linker thatis hybridized to the first adapter segment to the 5′ end of the miRNA ofinterest that is hybridized to the miRNA binding segment, and ligatingthe 3′ end of the miRNA of interest that is hybridized to the miRNAbinding segment to the 5′ end of the second linker that is hybridized tothe second adapter segment, thereby producing a complex defined as astrand of first linker, miRNA of interest and second linker that havebeen ligated together (ligated first linker-miRNA of interest-secondlinker) and that is hybridized to the capture probe; and g)dehybridizing the capture probe from the strand of the ligated firstlinker-miRNA of interest-second linker, where the miRNA of interest hasan miRNA of interest sequence, and comprises a 3′ end and a 5′ end,where the miRNA of interest is substantially complementary to, andcapable of hybridizing to, the miRNA binding segment of the captureprobe by Watson-Crick base pairing, where the first linker has a firstlinker sequence, and comprises a 3′ end and a 5′ end, where the firstlinker is substantially complementary to, and capable of hybridizing to,the first adapter segment of the capture probe by Watson-Crick basepairing, where the second linker has a second linker sequence, andcomprises a 3′ end and a 5′ end, and where the second linker issubstantially complementary to, and capable of hybridizing to, thesecond adapter segment of the capture probe by Watson-Crick basepairing. In another embodiment, the sample further comprises one or morethan one substance that is chemically related to the miRNA of interestselected from the group consisting of an RNA other than miRNA and a DNA.In another embodiment, the sample is from a eukaryote. In anotherembodiment, the sample is from a primate. In another embodiment, thesample is from a human. In another embodiment, the sample comprises atissue or fluid selected from the group consisting of blood, brain,heart, intestine, liver, lung, pancreas, muscle, a leaf, a flower, aplant root and a plant stem. In another embodiment, the miRNA ofinterest consists of 18 or 19 or 20 or 21 or 22 or 23 or 24 RNAresidues. In one embodiment, the miRNA of interest is listed in a publicdatabase. In another embodiment, the sample provided comprises aplurality of miRNAs of interest, and each of the plurality of miRNAs ofinterest have miRNA of interest sequences that are identical to oneanother. In another embodiment, the sample provided comprises aplurality of miRNAs of interest comprising a first miRNA of interesthaving a first miRNA of interest sequence, and a second miRNA ofinterest having a second miRNA of interest sequence, and where the firstmiRNA of interest sequence is different from the second miRNA ofinterest sequence. In another embodiment, the sample provided comprisesa plurality of miRNAs of interest comprising a first miRNA of interesthaving a first miRNA of interest sequence, a second miRNA of interesthaving a second miRNA of interest sequence, and a third miRNA ofinterest having a third miRNA of interest sequence, where the firstmiRNA of interest sequence is different from the second miRNA ofinterest sequence, where the first miRNA of interest sequence isdifferent from the third miRNA of interest sequence, and where secondmiRNA of interest sequence is different from the third miRNA of interestsequence. In another embodiment, the method further comprises isolatingthe total RNA from the sample after providing the sample.

In one embodiment, the capture probe provided is a set of captureprobes, where each of the capture probes comprises identical firstadapter segment sequences, where each of the capture probes of the setof capture probes comprises identical miRNA binding segment sequences,and where each of the capture probes of the set of capture probescomprises identical second adapter segment sequences. In anotherembodiment, the capture probe provided is a set of capture probes, wherethe set comprises at least one capture probe comprising an miRNA bindingsegment that is substantially complementary to, and capable ofhybridizing to, each miRNA listed in a single public database. Inanother embodiment, the capture probe provided is a set of captureprobes, where the set comprises a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical first adapter segment sequences, where the firstcapture probe and the second capture probe have identical miRNA bindingsegment sequences, and where the first capture probe has a secondadapter segment sequence that is different from the second adaptersegment sequence of the second capture probe. In another embodiment, thecapture probe provided is a set of capture probes, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical firstadapter segment sequences, where the first capture probe and the secondcapture probe have identical second adapter segment sequences, and wherethe first capture probe has an miRNA binding segment sequence that isdifferent from the miRNA binding segment sequence of the second captureprobe. In another embodiment, the capture probe provided is a set ofcapture probes, where the set comprises a first capture probe and asecond capture probe, where the first capture probe and the secondcapture probe have identical miRNA binding segment sequences, where thefirst capture probe and the second capture probe have identical secondadapter segment sequences, and where the first capture probe has a firstadapter segment sequence that is different from the first adaptersegment sequence of the second capture probe. In another embodiment, thecapture probe provided is a set of capture probes, where the setcomprises a first capture probe and a second capture probe, where thefirst capture probe and the second capture probe have identical firstadapter segment sequences, where the first capture probe has an miRNAbinding segment sequence that is different from the miRNA bindingsegment sequence of the second capture probe, and where the firstcapture probe has a second adapter segment sequence that is differentfrom the second adapter segment sequence of the second capture probe. Inanother embodiment, the capture probe provided is a set of captureprobes, where the set comprises a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical miRNA binding segment sequences, where the firstcapture probe has a first adapter segment sequence that is differentfrom the first adapter segment sequence of the second capture probe, andwhere the first capture probe has a second adapter segment sequence thatis different from the second adapter segment sequence of the secondcapture probe. In another embodiment, the capture probe provided is aset of capture probes, where the set comprises a first capture probe anda second capture probe, where the first capture probe and the secondcapture probe have identical second adapter segment sequences, where thefirst capture probe has a first adapter segment sequence that isdifferent from the first adapter segment sequence of the second captureprobe, and where the first capture probe has an miRNA binding segmentsequence that is different from the miRNA binding segment sequence ofthe second capture probe. In another embodiment, the capture probeprovided is a set of capture probes, where the set comprises a firstcapture probe and a second capture probe, where the first capture probehas a first adapter segment sequence that is different from the firstadapter segment sequence of the second capture probe, where the firstcapture probe has an miRNA binding segment sequence that is differentfrom the miRNA binding segment sequence of the second capture probe, andwhere the first capture probe has an miRNA binding segment sequence thatis different from the miRNA binding segment sequence of the secondcapture probe. In another embodiment, the capture probe provided is aset of capture probes, where the set comprises a first capture probehaving a first capture probe sequence, a second capture probe having asecond capture probe sequence, and a third capture probe having a thirdcapture probe sequence, where the first capture probe sequence isdifferent from the second capture probe sequence, where the firstcapture probe sequence is different from the third capture probesequence, and where second capture probe sequence is different from thethird capture probe sequence.

In one embodiment, the first linker segment and the second linkersegment comprise a substance selected from the group consisting of oneor more than one type of polynucleotide, one or more than one type ofpolynucleotide analog, and a combination of one or more than one type ofpolynucleotide and polynucleotide analog. In another embodiment, thefirst linker, or the second linker, or both the first linker and thesecond linker are resistant to nuclease degradation. In anotherembodiment, the first linker, or the second linker, or both the firstlinker and the second linker comprise nuclease resistant nucleotides. Inanother embodiment, the first linker, or the second linker, or both thefirst linker and the second linker comprise nucleotides with aphosphothioate backbone that render the first linker, or the secondlinker, or both the first linker and the second linker resistant tonuclease degradation. In another embodiment, the first linker, or thesecond linker, or both the first linker and the second linker comprisenuclease resistant nucleotides and comprise nucleotides with aphosphothioate backbone that render the first linker, or the secondlinker, or both the first linker and the second linker resistant tonuclease degradation. In another embodiment, the first linker and thesecond linker, each comprises between 6 and 50 residues. In anotherembodiment, the first linker comprises at least 10 residues, and atleast 10 residues at the 3′ end of the first linker are exactly thecomplement of the corresponding residues at or near the 5′ end of thefirst adapter segment. In another embodiment, the second linkercomprises at least 10 residues, and at least 10 residues at the 5′ endof the second linker are exactly the complement of the correspondingresidues at or near the 3′ end of the second adapter segment. In anotherembodiment, the 5′ end of the first linker, or the 3′ end of the secondlinker, or both the 5′ end of the first linker and the 3′ end of thesecond linker comprise a label. In another embodiment, the 5′ end offirst linker comprises one or more than one residue that extends beyondthe 3′ end of the first adapter segment after the first linkerhybridizes to the first adapter segment. In one embodiment, the one ormore than one residue of the 5′ end of first linker that extends beyondthe 3′ end of the first adapter segment functions as a primer bindingsite. In another embodiment, the 3′ end of second linker comprises oneor more than one residue that extends beyond the 5′ end of the secondadapter segment after the second linker hybridizes to the second adaptersegment. In one embodiment, the one or more than one residue of the 3′end of second linker that extends beyond the 5′ end of the secondadapter segment functions as a primer binding site. In anotherembodiment, the sample, the capture probe, the first linker and thesecond linker are combined simultaneously.

In one embodiment, the method further comprises adding one or more thanone RNAse inhibitor to the combination of the sample, the capture probe,the first linker and the second linker. In another embodiment, the firstadapter segment comprises a solid phase binding group, or the secondadapter segment comprises a solid phase binding group, or both the firstadapter segment comprises a solid phase binding group and the secondadapter segment comprises a solid phase binding group, and the methodfurther comprises binding the capture probe to a solid phase before orafter combining the sample, the capture probe, the first linker and thesecond linker. In another embodiment, the solid phase is a plurality ofparamagnetic particles. In another embodiment, the capture probe isbound to a solid phase through the first adapter segment or through thesecond adapter segment or through both the first adapter segment and thesecond adapter segment, and the method further comprises purifying thecapture probes with hybridized first linker, miRNA of interest andsecond linker—bound to the solid phase by removing non-hybridized firstlinkers, second linkers and any other substances that are not bound tothe solid phase. In another embodiment, the solid phase is contained ina vessel comprising a surface and a cap, and purifying comprisesapplying a magnetic field to attract the solid phase to the surface ofthe vessel or the cap of the vessel. In another embodiment, the firstlinker hybridizes to the first adapter segment at a position where thelast residue on the 3′ end of the first linker hybridizes to a residueon the first adapter segment that is between 1 residue and 5 residuesfrom the 3′ end of the miRNA binding segment. In another embodiment, thefirst linker hybridizes to the first adapter segment at a position wherethe last residue on the 3′ end of the first linker hybridizes to aresidue on the first adapter segment that is immediately adjacent to the3′ end of the miRNA binding segment. In another embodiment, the secondlinker hybridizes to the second adapter segment at a position where thelast residue on the 5′ end of the second linker hybridizes to a residueon the second adapter segment that is between 1 residue and 5 residuesfrom the 5′ end of the miRNA binding segment. In another embodiment, thesecond linker hybridizes to the second adapter segment at a positionwhere the last residue on the 5′ end of the second linker hybridizes toa residue on the second adapter segment that is immediately adjacent tothe 5′ end of the miRNA binding segment. In another embodiment, themethod further comprises purifying the complex. In another embodiment,the complex is bound to a solid phase through the first adapter segmentor through the second adapter segment or through both the first adaptersegment and the second adapter segment, and the method further comprisespurifying the complex by removing non-hybridized first linkers, secondlinkers and any other substances that are not bound to the solid phase.In another embodiment, the method further comprises purifying theligated first linker-miRNA of interest-second linker that has beendehybridized from the capture probe.

In one embodiment, the first linker, or the second linker, or both thefirst linker and the second linker comprise nuclease resistantnucleotides, or comprise nucleotides with a phosphothioate backbone thatrender the first linker, or the second linker, or both the first linkerand the second linker resistant to nuclease degradation, and purifyingthe ligated first linker-miRNA of interest-second linker comprisesapplying DNAase to a solution containing the ligated first linker-miRNAof interest-second linker to destroy any DNA present in the solution. Inanother embodiment, purifying the ligated first linker-miRNA ofinterest-second linker comprises circularizing the ligated firstlinker-miRNA of interest-second linker.

According to another embodiment, of the present invention, there isprovided a method for identifying an miRNA of interest. The methodcomprises: a) isolating the miRNAs according to the present invention,and b) sequencing the miRNA of interest portion of the strand of theligated first linker-miRNA of interest-second linker. In one embodiment,sequencing comprises subjecting the strand of the ligated firstlinker-miRNA of interest-second linker to reverse transcription toproduce a double stranded product comprising a first strand of theligated first linker-miRNA of interest-second linker and a second strandthat is the complement of the first strand. In another embodiment,sequencing comprises amplifying the double stranded product to produceamplification products. In another embodiment, sequencing comprisescloning the amplification products and culturing the amplificationproducts.

FIGURES

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures where:

FIG. 1 is a schematic diagram of a capture probe according to thepresent invention;

FIG. 2 through FIG. 6 show diagrams of some of the steps in certainembodiments of a method for isolating miRNAs and the method foridentifying miRNAs, according to the present invention; and

FIG. 7 shows a sequence trace of the miRNA isolated according to thepresent invention compared to a reference sequence of human miRNA.

DESCRIPTION

According to one embodiment of the present invention, there is provideda method for isolating microRNAs (miRNAs). According to anotherembodiment of the present invention, there is provided a method foridentifying miRNAs. In one embodiment, the method for identifying miRNAscomprises, first, isolating the miRNAs according to the presentinvention. According to another embodiment of the present invention,there is provided one or more than one capture probe and one or morethan one set of capture probes, suitable for use with a method forisolating miRNAs. In one embodiment, the method for isolating miRNAs isa method according to the present invention. The method and captureprobes will now be disclosed in detail.

As used in this disclosure, except where the context requires otherwise,the term “comprise” and variations of the term, such as “comprising,”“comprises” and “comprised” are not intended to exclude other additives,components, integers or steps.

As used in this disclosure, the term “miRNAs” means a naturallyoccurring, single stranded polyribonucleotide (polyRNA) of between 18and 24 RNA residues, which is derived from a larger, naturally occurringnoncoding eukaryotic precursor RNA (usually having a ‘hairpin’configuration).

As used in this disclosure, the terms “one or more than one miRNAs,” “anmiRNA” and “the miRNA” are intended to be synonymous, that is, areintended to indicate either one miRNA of interest or a plurality ofmiRNA of interest, except where the context requires otherwise.

As used in this disclosure, the terms “one or more than one captureprobe,” “a capture probe,” “the capture probe” and “the capture probes”are intended to be synonymous, that is, are intended to indicate eitherone capture probe or a plurality of capture probes, except where thecontext requires otherwise.

As used in this disclosure, the terms “a first linker,” “the firstlinker” and “the first linkers” are intended to be synonymous, that is,are intended to indicate either one first linker or a plurality of firstlinkers, except where the context requires otherwise.

As used in this disclosure, the terms “a second linker,” “the secondlinker” and “the second linkers” are intended to be synonymous, that is,are intended to indicate either one second linker or a plurality ofsecond linkers, except where the context requires otherwise.

As used in this disclosure, the term “substantially complementary” andvariations of the term, such as “substantial complement,” means that atleast 90% of all of the consecutive residues in a first strand arecomplementary to a series of consecutive residues of the same length ofa second strand. As will be understood by those with skill in the artwith reference to this disclosure, one strand can be shorter than theother strand and still be substantially complementary. With respect tothe invention disclosed in this disclosure, for example, the firstadapter segment can be shorter than the first linker and still besubstantially complementary to the first linker, and the second adaptersegment can be shorter than the second linker and still be substantiallycomplementary. The miRNA binding segment can be the same length orlonger than the miRNA of interest.

As used in this disclosure, the term “hybridize” and variations of theterm, such as “hybridizes” and “hybridized,” means a Watson-Crick basepairing of complementary nucleic acid single strands or segments ofstrands to produce, an anti-parallel, double-stranded nucleic acid, andas used in this disclosure, hybridization should be understood to bebetween substantially complementary strands unless specified otherwise,or where the context requires otherwise. As an example, hybridizationcan be accomplished by combining equal molar concentrations of each ofthe pairs of single strands, such as 100 pmoles, in the presence of 5 ugyeast tRNA in a total volume of 50 μl of aqueous buffer containing 400mM MOPS, 80 mM DTT, and 40 mM MgCl₂ at a pH of 7.3, and then incubatingthe mixture at 25° C. for one hour while shaking gently.

As used in this disclosure, the term “near the end” and variations ofthe term, means within 20% of the residues of the identified endresidue. For example, near the end of a 20 residue strand, means thefirst four residues of the identified end of the strand.

According to one embodiment of the present invention, there is provideda capture probe suitable for use with a method for isolating miRNAsaccording to the present invention. Referring now to FIG. 1, there isshown a schematic diagram of a capture probe 10 according to oneembodiment of the present invention. The capture probe 10, and each ofits segments, comprises a substance selected from the group consistingof one or more than one type of polynucleotide, includingribonucleotides and deoxynucleotides, one or more than one type ofpolynucleotide analog, and a combination of one or more than one type ofpolynucleotide and polynucleotide analog. As can be seen in FIG. 1, thecapture probe 10 comprises three segments: a) a first adapter segment 12having a first adapter segment sequence, b) a second adapter segment 14having a second adapter segment sequence, and c) an miRNA bindingsegment 16 having an miRNA binding segment sequence, where the miRNAbinding segment 16 is between the first adapter segment 12 and thesecond adapter segment 14.

According to one embodiment of the present invention, there is provideda set of capture probes comprising at least one capture probe comprisingan miRNA binding segment that is substantially complementary to, andcapable of hybridizing to, each miRNA listed M a single public database.

According to one embodiment of the present invention, there is provideda plurality of capture probes, where each capture probe of the pluralityof capture probes comprises identical first adapter segment sequences,where each capture probe of the plurality of capture probes comprisesidentical miRNA binding segment sequences, and where each capture probeof the plurality of capture probes comprises identical second adaptersegment sequences.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical first adapter segment sequences, where the firstcapture probe and the second capture probe have identical miRNA bindingsegment sequences, and where the first capture probe has a secondadapter segment sequence that is different from the second adaptersegment sequence of the second capture probe.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical first adapter segment sequences, where the firstcapture probe and the second capture probe have identical second adaptersegment sequences, and where the first capture probe has an miRNAbinding segment sequence that is different from the miRNA bindingsegment sequence of the second capture probe.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical miRNA binding segment sequences, where the firstcapture probe and the second capture probe have identical second adaptersegment sequences, and where the first capture probe has a first adaptersegment sequence that is different from the first adapter segmentsequence of the second capture probe.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical first adapter segment sequences, and where thefirst capture probe has an miRNA binding segment sequence that isdifferent from the miRNA binding segment sequence of the second captureprobe, and where the first capture probe has a second adapter segmentsequence that is different from the second adapter segment sequence ofthe second capture probe.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical miRNA binding segment sequences, where the firstcapture probe has a first adapter segment sequence that is differentfrom the first adapter segment sequence of the second capture probe, andwhere the first capture probe has a second adapter segment sequence thatis different from the second adapter segment sequence of the secondcapture probe.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe and the second captureprobe have identical second adapter segment sequences, where the firstcapture probe has a first adapter segment sequence that is differentfrom the first adapter segment sequence of the second capture probe, andwhere the first capture probe has an miRNA binding segment sequence thatis different from the miRNA binding segment sequence of the secondcapture probe.

According to one embodiment of the present invention, there is provideda set of capture probes comprising a first capture probe and a secondcapture probe, where the first capture probe has a first adapter segmentsequence that is different from the first adapter segment sequence ofthe second capture probe, where the first capture probe has an miRNAbinding segment sequence that is different from the miRNA bindingsegment sequence of the second capture probe, and where the firstcapture probe has a second adapter segment sequence that is differentfrom the second adapter segment sequence of the second capture probe.

Referring again to FIG. 1, the first adapter segment 12 comprises a 3′end 18 and a 5′ end 20. As can be seen in FIG. 1, the 5′ end 20 of thefirst adapter segment 12 is connected to the 3′ end 22 of the miRNAbinding segment 16, that is the first adapter segment 12 is connectedupstream of the miRNA binding segment 16. In one embodiment, the firstadapter segment 12 is substantially complementary to and capable ofhybridizing a first linker probe designated in this disclosure as a“first linker.” When used in the method of the present invention, thefirst adapter segment 12 facilitates the ligation of the 3′ end of thefirst linker to the 5′ end of the miRNA of interest by aligning thefirst linker in position for ligation to the miRNA of interest.

In one embodiment, the first adapter segment has a number of residuesbetween 5 and 50. In another embodiment, the first adapter segment has anumber of residues between 5 and 20. In another embodiment, the firstadapter segment has a number of residues between 6 and 16.

In one embodiment, the first adapter segment 12 comprises one or morethan one sequence 24 or sequence 26 that is a restriction site motif. Ina particularly preferred embodiment, the specific restriction sitemotif, when present, is not present in the DNA analog of the miRNA ofinterest that is being isolated and identified by the present method. Inone embodiment, the restriction site motif is acted upon by arestriction enzyme selected from the group consisting of BamH I, HindIII and EcoR I. In a preferred embodiment, the restriction site motif isacted upon by a restriction enzyme selected from the group consisting ofNot I, Xho I, Xma I and Nhe I, because BamH I, Hind III and EcoR I alsoact upon some sequences of miRNA. As will be understood by those withskill in the art with reference to this disclosure, however, othersuitable restriction site motifs can also be used.

In another embodiment, the first adapter segment 12 comprises a sequence24 or a sequence 26 that is a polynucleotide synthesis promoter motiffor a polynucleotide polymerase, or that is complementary to apolynucleotide synthesis promoter motif for a polynucleotide polymerase.In a preferred embodiment, the polynucleotide synthesis promoter motifis a motif for a polynucleotide synthesis promoter selected from thegroup consisting of T7, SP6, a T3 DNA dependent RNA polymerase, a type 2RNA polymerase of E. coli and single stranded DNA dependent N4 RNApolymerase. The polynucleotide synthesis promoter motif can be a motiffor any other suitable polynucleotide synthesis promoter, however, aswill be understood by those with skill in the art with reference to thisdisclosure.

As will be understood by those with skill in the art with reference tothis disclosure, the sequence that is a restriction site motif of thefirst adapter segment 12 can be in either position 24 or in the position26 as indicated in FIG. 1, and the sequence that is a polynucleotidesynthesis promoter motif can be in either position 24 or in the position26 as indicated in FIG. 1. In a preferred embodiment, there is no othera restriction site motif sequence of the first adapter segment 12 otherthan in the position 24 or in the position 26 as shown in FIG. 1.

In another embodiment, the first adapter segment 12 comprises a solidphase binding group 28 to immobilize the capture probe 10 to a solidphase. In one embodiment, the solid phase binding group 28 is at or nearthe 3′ end 18 of the first adapter segment 12, however, as will beunderstood by those with skill in the art with reference to thisdisclosure, the solid phase binding group 28 can be anywhere on thecapture probe 10 other than at or near the 3′ end 18 of the firstadapter segment 12. In one embodiment, the solid phase binding group 28immobilizes the capture probe 10 to a solid phase covalently. In anotherembodiment, the solid phase binding group 28 immobilizes the captureprobe 10 to a solid phase non-covalently. In one embodiment, the solidphase binding group 28 immobilizes the capture probe 10 to a solid phasereversibly. As used in this context, “reversibly” means that the solidphase binding group 28 immobilizes the capture probe 10 to a solid phasein such a way that the solid phase binding group 28 can be disassociatedfrom the solid phase without destruction of the capture probe 10 andwithout disruption of hybridization between the capture probe 10 and theligated first linker 48-miRNA of interest 42-second linker 50 (asdisclosed below). In another embodiment, the solid phase binding group28 immobilizes the capture probe 10 to a solid phase non-reversibly. Forexample, in one embodiment, the solid phase binding group 28 immobilizesthe capture probe 10 to a solid phase non-covalently and reversibly,where the solid phase binding group 28 comprises biotin or an analog ofbiotin capable of binding with avidin or streptavidin or functionalanalogs of avidin or streptavidin with high affinity, such as with anaffinity having an affinity constant of between about 10^(e12) and10^(e20). Additionally for example, in one embodiment the solid phasebinding group 28 of the first adapter segment 12 immobilizes the captureprobe 10 to a solid phase covalently and non-reversibly, where the solidphase binding group 28 comprises a terminal 5′ primary amino group atthe 3′ end 18 of the first adapter segment 12 for coupling to a solidphase surface having free carboxyl groups using standard carbodiimidechemistry, as will be understood by those with skill in the art withreference to this disclosure. Further, as will be understood by thosewith skill in the art with reference to this disclosure, any solid phasebinding group 28 present in the first adapter segment 12, and anytechnique for coupling the solid phase binding group 28 to a solid phaseused in connection with the present method should not interfere with thehybridization and capture of the miRNA of interest to the miRNA bindingsegment 16, or with any other step of the present method.

By way of example only, in one embodiment the first adapter segment 12comprises DNA and has a first adapter segment sequence in the 5′ to 3′direction of ATTTAGGTGACACTATAG, SEQ ID NO:1.

The second adapter segment 14 comprises a 3′ end 30 and a 5′ end 32. Ascan be seen in FIG. 1, the 3′ end 30 of the second adapter segment 14 isconnected to the 5′ end 34 of the miRNA binding segment 16, that is thesecond adapter segment 14 is connected downstream of the miRNA bindingsegment 16. In one embodiment, the second adapter segment 14 issubstantially complementary to and capable of hybridizing a secondlinker probe designated in this disclosure as a “second linker.” Whenused in the method of the present invention, the second adapter segment14 facilitates the ligation of the 5′ end of the second linker to the 3′end of the miRNA of interest by aligning the second linker in positionfor ligation to the miRNA of interest.

In one embodiment, the second adapter segment 14 has a number ofresidues between 5 and 50. In another embodiment, the second adaptersegment 14 has a number of residues between 5 and 20. In anotherembodiment, the second adapter segment 14 has a number of residuesbetween 6 and 16.

In another embodiment, the second adapter segment 14 comprises one ormore than one sequence 36 that is a restriction site motif. In aparticularly preferred embodiment, the specific restriction site motif,when present, is not present in the DNA analog of the miRNA of interestthat is being isolated and identified by the present methods. In oneembodiment, the restriction site motif is acted upon by a restrictionenzyme selected from the group consisting of BamH I, Hind III and EcoRI. In a preferred embodiment, the restriction site motif is acted uponby a restriction enzyme selected from the group consisting of Not I, XhoI, Xma I and Nhe I, because BamH I, Hind III and EcoR I also act uponsome sequences of miRNA. As will be understood by those with skill inthe art with reference to this disclosure, however, other suitablerestriction site motifs can also be used.

In one embodiment, the one or more than one sequence 24 that is arestriction site motif is identical to the one or more than one sequence36 that is a restriction site motif. In another embodiment, the one ormore than one sequence 24 that is a restriction site motif is differentfrom the one or more than one sequence 36 that is a restriction sitemotif.

In one embodiment, the second adapter segment 14 comprises a sequence 38that is a polynucleotide synthesis promoter motif for a polynucleotidepolymerase, or that is complementary to a polynucleotide synthesispromoter motif for a polynucleotide polymerase. In a preferredembodiment, the polynucleotide synthesis promoter motif is a motif for apolynucleotide synthesis promoter selected from the group consisting ofT7, SP6, a T3 DNA dependent RNA polymerase, a type 2 RNA polymerase ofE. coli and single stranded DNA dependent N4 RNA polymerase. Thepolynucleotide synthesis promoter motif can be a motif for any othersuitable polynucleotide synthesis promoter, however, as will beunderstood by those with skill in the art with reference to thisdisclosure.

As will be understood by those with skill in the art with reference tothis disclosure, the sequence that is a restriction site motif of thesecond adapter segment 14 can be in either position 36 or in theposition 38 as indicated in FIG. 1, and the sequence that is apolynucleotide synthesis promoter motif can be in either position 36 orin the position 38 as indicated in FIG. 1. In a preferred embodiment,there is no other a restriction site motif sequence of the secondadapter segment 14 other than in the position 36 or in the position 38as shown in FIG. 1.

By way of example only, in one embodiment the second adapter segment 14comprises DNA and has a second adapter segment sequence in the 5′ to 3′direction of CCCTATAGTGAGTCGTATTA SEQ ID NO:2.

In another embodiment, the second adapter segment 14 comprises a solidphase binding group 40 to immobilize the capture probe 10 to a solidphase. In one embodiment, the solid phase binding group 40 is at or nearthe 5′ end 32 of the second adapter segment 14, however, as will beunderstood by those with skill in the art with reference to thisdisclosure, the solid phase binding group 40 can be anywhere on thecapture probe 10 other than at or near the 5′ end 32 of the secondadapter segment 14. In one embodiment, the solid phase binding group 40immobilizes the capture probe 10 to a solid phase covalently. In anotherembodiment, the solid phase binding group 40 immobilizes the captureprobe 10 to a solid phase non-covalently. In one embodiment, the solidphase binding group 40 immobilizes the capture probe 10 to a solid phasereversibly. As used in this context, “reversibly” means that the solidphase binding group 40 immobilizes the capture probe 10 to a solid phasein such a way that the solid phase binding group 40 can be disassociatedfrom the solid phase without destruction of the capture probe 10 andwithout disruption of hybridization between the capture probe 10 and theligated first linker 48-miRNA of interest 42-second linker 50 (asdisclosed below). In another embodiment, the solid phase binding group40 immobilizes the capture probe 10 to a solid phase non-reversibly. Forexample, in one embodiment, the solid phase binding group 40 immobilizesthe capture probe 10 to a solid phase non-covalently and reversibly,where the solid phase binding group 40 comprises biotin or an analog ofbiotin capable of binding with avidin or streptavidin or functionalanalogs of avidin or streptavidin with high affinity, such as with anaffinity having an affinity constant of between about 10^(e12) and10^(e20). Additionally for example, in one embodiment the solid phasebinding group 40 of the second adapter segment 14 immobilizes thecapture probe 10 to a solid phase covalently and non-reversibly, wherethe solid phase binding group 40 comprises a terminal 3′ primary aminogroup at the 5′ end 32 of the second adapter segment 14 for coupling toa solid phase surface having free carboxyl groups using standardcarbodiimide chemistry, as will be understood by those with skill in theart with reference to this disclosure. Further, as will be understood bythose with skill in the art with reference to this disclosure, any solidphase binding group 40 present in the second adapter segment 14, and anytechnique for coupling the solid phase binding group 40 to a solid phaseused in connection with the present method should not interfere with thehybridization and capture of the miRNA of interest to the miRNA bindingsegment 16, or with any other step of the present method.

In another embodiment, both the first adapter segment 12 comprises asolid phase binding group 28, and the second adapter segment 14comprises a solid phase binding group 40. In another embodiment, boththe first adapter segment 12 comprises a solid phase binding group 28 ator near the 3′ end 18 of the first adapter segment 12, and the secondadapter segment 14 comprises a solid phase binding group 40 at or nearthe 5′ end 32 of the second adapter segment 14.

Referring again to FIG. 1 and as stated above, the capture probe 10 ofthe present invention further comprises an miRNA binding segment 16. ThemiRNA binding segment 16 has an miRNA binding segment sequencecomprising a 3′ end 22 and a 5′ end 34, and consists of one or more thanone type of polynucleotide, including ribonucleotides anddeoxynucleotides, or one or more than one type of polynucleotide analog,or a combination of one or more than one type of polynucleotide andpolynucleotide analog. The 3′ end 22 of the miRNA binding segment 16 isconnected to the 5′ end 20 of the first adapter segment 12 of thecapture probe 10 according to the present invention, that is, the firstadapter segment 12 is connected upstream of the miRNA binding segment16. The 5′ end 34 of the miRNA binding segment 16 is connected to the 3′end 30 of the second adapter segment 14 of the capture probe 10according to the present invention, that is, the second adapter segment14 is connected downstream of the miRNA binding segment 16.

In one embodiment, the miRNA binding segment consists of between 18 and24 DNA residues. In another embodiment, the miRNA binding segment 16consists of 18 or 19 or 20 or 21 or 22 or 23 or 24 residues selectedfrom the group consisting of DNA, RNA, chimeric DNA/RNA, DNA analogs andRNA analogs.

The miRNA binding segment 16 is substantially complementary to, andcapable of hybridizing to, one or more than one miRNA of interest byWatson-Crick base pairing, including an miRNA of interest having apredetermined sequence or having a predetermined size, from a sample. Inone embodiment, the sample comprises substances that are chemicallyrelated, such as for example, a mixture of messenger RNAs, transferRNAs, ribosomal RNAs and genomic DNA. An miRNA of interest can beselected from any known miRNAs from any suitable source, as will beunderstood by those with skill in the art with reference to thisdisclosure. In one embodiment, the miRNA of interest is selected from apublic database. In a preferred embodiment, the central repositoryprovided is the Sanger Institute http://microrna.sanger.ac.uk/sequences/to which newly discovered and previously known miRNA sequences can besubmitted for naming and nomenclature assignment, as well as placementof the sequences in a database for archiving and for online retrievalvia the world wide web. Generally, the data collected on the sequencesof miRNAs by the Sanger Institute include species, source, correspondinggenomic sequences and genomic location (usually chromosomalcoordinates), as well as full length transcription products andsequences for the mature fully processed miRNA (miRNA with a 5′ terminalphosphate group).

To select the sequence or sequences of the miRNA binding segment 16, anmiRNA of interest, or set of miRNAs of interest is selected from asuitable source, such as for example, the Sanger Institute database orother suitable database, as will be understood by those with skill inthe art with reference to this disclosure. If a set of miRNAs ofinterest is selected from one or more than one source that containsduplicate entries for one or more than one miRNA, in a preferredembodiment, the duplicated entries are first removed so that the set ofsequences of miRNAs of interest contains only one sequence for eachmiRNA of interest. In one embodiment, the set of miRNAs of interestconsists of one of each miRNA from a single source or database,including a public source or public database, such as one of each miRNAlisted in the central repository provided by the Sanger Institute.

In another embodiment the miRNA of interest is a eucaryotic miRNA. Inanother embodiment the miRNA of interest is a primate miRNA. In apreferred embodiment, the miRNA of interest is a human miRNA. In anotherembodiment, the miRNAs in the set of miRNAs of interest are alleucaryotic miRNAs. In another embodiment, the miRNAs in the set ofmiRNAs of interest are all primate miRNAs. In another embodiment, themiRNAs in the set of miRNAs of interest are all human miRNAs.

Next, the miRNA binding segment is selected to be the substantialcomplement of the miRNA of interest sequence. In a preferred embodiment,the miRNA binding segment is the exact complement to the miRNA ofinterest in both length and sequence. In another embodiment, the miRNAbinding segment is more than 90% complementary to a segment of the miRNAof interest of the same length as the miRNA of interest sequence. Inanother embodiment, the miRNA binding segment is more than 80%complementary to a segment of the miRNA of interest of the same lengthas the miRNA of interest sequence.

In one embodiment, the miRNA binding segment 16 consists of RNA. In oneembodiment, the miRNA binding segment 16 consists of DNA. In oneembodiment, the miRNA binding segment 16 consists of polynucleotideanalogs. In one embodiment, the miRNA binding segment 16 consists of achimera of more than one polynucleotide or polynucleotide analogselected from the group consisting of RNA, DNA, polynucleotide analogsof RNA, and polynucleotide analogs of DNA. Once, the miRNA bindingsegment sequence is selected, the miRNA binding segment 16 issynthesized according to standard synthesis techniques known to thosewith skill in the art, as will be understood by those with skill in theart with reference to this disclosure.

Table I provides a list of ten sample miRNA binding segments 16 whichconsist of DNA along with the miRNAs that are the exact complement ofthe miRNA binding segments. As will be understood by those with skill inthe art with reference to this disclosure, and as disclosed in thisdisclosure, this is a sample list of miRNA binding segments 16, and anyother sequence serving the function of the miRNA binding segments willalso be useful, including for example miRNA binding segments 16 that arethe RNA of the miRNA binding segments 16 listed in Table I.

TABLE I miRNA that is miRNA binding complementary to segment sequencemiRNA binding SEQ ID NO: 5′-3′ segment SEQ ID NO: 3 AACTATACAACCTACTACChsa-let-7a TCA SEQ ID NO: 4 AACCACACAACCTACTACC hsa-let-7b TCASEQ ID NO: 5 AACCATACAACCTACTACC hsa-let-7c TCA SEQ ID NO: 6ACTATGCAACCTACTACCT hsa-let-7d CT SEQ ID NO: 7 ACTATACAACCTCCTACCThsa-let-7e CA SEQ ID NO: 8 AACTATACAATCTACTACC hsa-let-7f TCASEQ ID NO: 9 ACTGTACAAACTACTACCT hsa-let-7g CA SEQ ID NO: 10ACAGCACAAACTACTACCT hsa-let-7i CA SEQ ID NO: 11 CACAAGTTCGGATCTACGGhsa-miR-100 GTT SEQ ID NO: 12 CTTCAGTTATCACAGTACT hsa-miR-101 GTA

Therefore, by way of example only, a capture probe 10 according to thepresent invention for use in a method for isolating miRNA hsa-let-7a,can have the following sequence in the 5′ to 3′ direction:

SEQ ID NO: 13 ATTTAGGTGACACTATAGAAACTATACAACCTACTACCTCACCCTATAGTGAGTCGTATTA,. 

According to one embodiment of the present invention, there is a set ofcapture probes 10 suitable for use with a method for isolating miRNAs.Referring now to Table II, in one embodiment, by way of example, the setconsists of at least seven capture probes 10 according to the presentinvention, where each capture probe 10 has a first adapter segment 12ATTTAGGTGACACTATAG, SEQ ID NO:1, a second adapter segment 14 ofCCCTATAGTGAGTCGTATTA SEQ ID NO:2, and an miRNA binding segment 16varying from 18 mer to 24 mer, and having a nucleotide or nucleotideanalog (N) (such as for example A, G, C, T as ribonucleotides ordeoxynucleotides) capable of hybridizing with a nucleotide on an miRNA.In a preferred embodiment, as shown, the 5′ end 32 of the second adaptersegment 14 is biotinylated to bind to a solid phase.

TABLE II Size of Capture Probe Sequence miRNA SEQ ID NO: 5′-3′ CapturedSEQ ID NO: 14 5′biotin- 18 mer ATTTAGGTGACACTATAGNNNNNNNNNNNNNNNNNNCCCTATAGTGAGTCGTATTA SEQ ID NO: 15 5′biotin- 19 merATTTAGGTGACACTATAGNNNNNNNNNN NNNNNNNNNCCCTATAGTGAGTCGTATT ASEQ ID NO: 16 5′biotin- 20 mer ATTTAGGTGACACTATAGNNNNNNNNNNNNNNNNNNNNCCCTATAGTGAGTCGTAT TA SEQ ID NO: 17 5′biotin- 21 merATTTAGGTGACACTATAGNNNNNNNNNN NNNNNNNNNNNCCCTATAGTGAGTCGTA TTASEQ ID NO: 18 5′biotin- 22 mer ATTTAGGTGACACTATAGNNNNNNNNNNNNNNNNNNNNNNCCCTATAGTGAGTCGT ATTA SEQ ID NO: 19 5′biotin- 23 merATTTAGGTGACACTATAGNNNNNNNNNN NNNNNNNNNNNNNCCCTATAGTGAGTCG TATTASEQ ID NO: 20 5′biotin- 24 mer ATTTAGGTGACACTATAGNNNNNNNNNNNNNNNNNNNNNNNNCCCTATAGTGAGTC GTATTA

The capture probe 10 of the present invention can be synthesizedaccording to standard techniques, as will be understood by those withskill in the art with reference to this disclosure. In one embodiment,the capture probe 10 is synthesized as a contiguous single sequence foreach miRNA of interest to be isolated and detected. In a preferredembodiment, there is provided a set of capture probes 10 comprising afirst capture probe and a second capture probe that are synthesizedseparately, where the sequence of the first capture probe has one ormore than one difference with the sequence of the second capture probe,and where the set of capture probes 10 is produced by mixing the firstcapture probe and the second capture probe after they are synthesized.

In one embodiment, the capture probes 10 are synthesized by combiningthe sequence text strings for the first adapter segment 12, the miRNAbinding segment 16, and the second adapter segment 14 in a database orspreadsheet to generate a capture probe 10 sequence, and thensynthesizing the capture probe 10 according to standard techniques, aswill be understood by those with skill in the art with reference to thisdisclosure. In one embodiment, the capture probes 10 are designed foruse in a method according to the present invention, and then purchasedfrom a vendor of polynucleotide or polynucleotide analog sequences, suchas for example, from Integrated DNA Technologies (Coralville, Iowa US)or Invitrogen Corp. (Carlsbad, Calif. US).

According to another embodiment of the present invention, there isprovided a method for isolating an miRNA (microRNA) of interest from asample comprising the miRNA of interest. According to another embodimentof the present invention, there is provided a method for identifyingmiRNAs. In one embodiment, the method for identifying miRNAs comprises,first, isolating the miRNAs according to the present invention.Referring now to FIG. 2 through FIG. 6, there are shown some of thesteps in certain embodiments of the methods. The steps shown are notintended to be limiting nor are they intended to indicate that each stepdepicted is essential to the method, but instead are exemplary stepsonly.

As can be seen, the method comprises, first, providing a samplecomprising an miRNA of interest 42. In one embodiment, the samplefurther comprises one or more than one substance that is chemicallyrelated to the miRNA of interest 42, such as for example, a substanceselected from the group consisting of messenger RNA, transfer RNA,ribosomal RNA, siRNA, 5S/5.8SrRNA, genomic DNA and a combination of thepreceding. In one embodiment, the sample further comprises one or morethan one RNA other than miRNA, such as for example, a substance selectedfrom the group consisting of messenger RNA, transfer RNA, ribosomal RNA,siRNA, 5S/5.8SrRNA and a combination of the preceding. All of the RNA inthe sample, regardless of the type of RNA, constitutes the “total RNA”in the sample.

In one embodiment, the sample is from a eukaryote. In anotherembodiment, the sample is from a primate. In a preferred embodiment, thesample is from a human.

In one embodiment, the sample comprises a tissue or fluid selected fromthe group consisting of blood, brain, heart, intestine, liver, lung,pancreas, muscle, a leaf, a flower, a plant root and a plant stem.

The miRNA of interest 42 has an miRNA of interest sequence, andcomprises 3′ end 44 and a 5′ end 46. In one embodiment, the miRNA ofinterest consists of between 18 and 24 RNA residues. In anotherembodiment, the miRNA of interest consists of 18 or 19 or 20 or 21 or 22or 23 or 24 RNA residues.

The miRNA of interest 42 is substantially complementary to, and capableof hybridizing to, an miRNA binding segment 16 of a capture probe 10according to the present invention by Watson-Crick base pairing. In oneembodiment, the miRNA of interest 42 is listed in a public database. Ina preferred embodiment, the public database is a central repositoryprovided by the Sanger Institute http://microrna.sanger.ac.uk/sequences/to which miRNA sequences are submitted for naming and nomenclatureassignment, as well as placement of the sequences in a database forarchiving and for online retrieval via the world wide web. Generally,the data collected on the sequences of miRNAs by the Sanger Instituteinclude species, source, corresponding genomic sequences and genomiclocation (chromosomal coordinates), well as full length transcriptionproducts and sequences for the mature fully processed miRNA (miRNA witha 5′ terminal phosphate group).

In one embodiment, the sample provided comprises a plurality of miRNAsof interest 42, where each of the plurality of miRNAs of interest 42 hasmiRNA of interest sequences that are identical to one another. In oneembodiment, the sample provided comprises a plurality of miRNAs ofinterest 42, where at least two of the plurality of miRNAs of interest42 have miRNA of interest sequences that are different from one another.In one embodiment, the sample provided comprises a plurality of miRNAsof interest 42 comprising a first miRNA of interest having a first miRNAof interest sequence, and a second miRNA of interest having a secondmiRNA of interest sequence, where the first miRNA of interest sequenceis different from the second miRNA of interest sequence. In anotherembodiment, the sample provided comprises a plurality of miRNAs ofinterest 42 comprising a first miRNA of interest having a first miRNA ofinterest sequence, a second miRNA of interest having a second miRNA ofinterest sequence, and a third miRNA of interest having a third miRNA ofinterest sequence, where the first miRNA of interest sequence isdifferent from the second miRNA of interest sequence, where the firstmiRNA of interest sequence is different from the third miRNA of interestsequence, and where second miRNA of interest sequence is different fromthe third miRNA of interest sequence.

In one embodiment, the method further comprises isolating the total RNAfrom the sample after providing the sample. In one embodiment, isolatingthe total RNA is accomplished according to techniques well known tothose with skill in the art, such as for example using a commerciallyavailable kit for the isolation of total RNA available from Ambion, Inc.(Austin, Tex. US), Invitrogen Corp. and Qiagen, Inc. (Valencia, Calif.US), among others, as will be understood by those with skill in the artwith reference to this disclosure. As will be understood by those withskill in the art with reference to this disclosure, when the methodcomprises isolating the total RNA from the sample after providing thesample, the term “sample” means the isolated total RNA for the remainingsteps in the method.

Next, the method further comprises providing a capture probe 10. In oneembodiment, the capture probe 10 provided is a capture probe 10according to the present invention. When the capture probe 10 is acapture probe 10 according to the present invention, in all respects,the capture probe 10 provided has the characteristics and attributes asdisclosed for a capture probe 10 according to the present invention,some of which will be repeated hereafter for clarity. The capture probe10 has a capture probe sequence, and comprises three segments: a) afirst adapter segment 12 having a first adapter segment sequence, andcomprising a 3′ end 18 and a 5′ end 20; b) a second adapter segment 14having a second adapter segment sequence, and comprising a 3′ end 30 anda 5′ end 32; and c) an miRNA binding segment 16 having an miRNA bindingsegment sequence, and comprising a 3′ end 22 and a 5′ end 34, where the5′ end 20 of the first adapter segment 12 is connected to the 3′ end 22of the miRNA binding segment 16, and where the 5′ end of the miRNAbinding segment 34 is connected to the 3′ end 30 of the second adaptersegment 14. The specificity of the miRNA binding segment 16 to the miRNAof interest 42 allows the method to be used directly on a samplecontaining substances related to miRNA or on isolated total RNA withoutrequiring the specific separation of miRNAs from the sample or from thetotal RNA, such as for example by either gel purification orchromatographic purification, as necessary in prior art methods.

In one embodiment, the capture probe 10 provided is a set of captureprobes, where each of the set of capture probes provided have captureprobe sequences that are identical to one another. In one embodiment,the capture probe 10 provided is a set of capture probes, where at leasttwo capture probes of the set of capture probes have capture probesequences that are different from one another. In another embodiment,the capture probe 10 is a set of capture probes comprising a firstcapture probe having a first capture probe sequence, and a secondcapture probe having a second capture probe sequence, where the firstcapture probe sequence is different from the second capture probesequence. In another embodiment, the capture probe 10 provided is a setof capture probes comprising a first capture probe having a firstcapture probe sequence, a second capture probe having a second captureprobe sequence, and a third capture probe having a third capture probesequence, where the first capture probe sequence is different from thesecond capture probe sequence, where the first capture probe sequence isdifferent from the third capture probe sequence, and where secondcapture probe sequence is different from the third capture probesequence.

Then, the method comprises providing a first linker 48 and a secondlinker 50. In one embodiment, the first linker segment and the secondlinker segment comprise a substance selected from the group consistingof one or more than one type of polynucleotide, includingribonucleotides and deoxynucleotides, one or more than one type ofpolynucleotide analog, and a combination of one or more than one type ofpolynucleotide and polynucleotide analog. In one embodiment, the firstlinker 48, or the second linker 50, or both the first linker 48 and thesecond linker 50 are resistant to nuclease degradation. In a preferredembodiment, the first linker 48, or the second linker 50, or both thefirst linker 48 and the second linker 50 comprise nuclease resistantnucleotides. In another preferred embodiment, the first linker 48, orthe second linker 50, or both the first linker 48 and the second linker50 comprise nucleotides with a phosphothioate backbone that render thefirst linker 48, or the second linker 50, or both the first linker 48and the second linker 50 resistant to nuclease degradation. In anotherpreferred embodiment, the first linker 48, or the second linker 50, orboth the first linker 48 and the second linker 50 comprise both nucleaseresistant nucleotides and nucleotides with a phosphothioate backbonethat render the first linker 48, or the second linker 50, or both thefirst linker 48 and the second linker 50 resistant to nucleasedegradation.

The first linker 48 has a first linker sequence, and comprises a 3′ end52 and a 5′ end 54. The first linker 48 is substantially complementaryto, and capable of hybridizing to, the first adapter segment 12 of acapture probe 10 according to the present invention by Watson-Crick basepairing. The second linker 50 has a second linker sequence, andcomprises a 3′ end 56 and a 5′ end 58. The second linker 50 issubstantially complementary to, and capable of hybridizing to, thesecond adapter segment 14 of a capture probe 10 according to the presentinvention by Watson-Crick base pairing. The first linker 48 and thesecond linker 50 each comprises between 6 and 50 residues.

In a preferred embodiment, the first linker 48 comprises at least 10residues, and at least 10 residues at the 3′ end 52 of the first linker48 are exactly the complement of the corresponding residues at or nearthe 5′ end 20 of the first adapter segment 12. In another embodiment,the second linker 50 comprises at least 10 residues, and at least 10residues at the 5′ end 58 of the second linker 50 are exactly thecomplement of the corresponding residues at or near the 3′ end 30 of thesecond adapter segment 14.

In a preferred embodiment, the 3′ end 52 of the first linker 48 iscapable of being ligated to the 5′ end 46 of the miRNA of interest 42 bya suitable ligase, such as for example T4 polynucleotide ligase, or byanother suitable chemical reaction.

In a preferred embodiment, the 5′ end 58 of the second linker 50 is anucleotide with a 5′ pyrophosphate bond between it and its adjacent 5′end nucleotide of the second adapter segment 14 to allow ligation of the5′ end 58 of the second linker 50 to the 3′ end 44 of the miRNA ofinterest 42 by a suitable ligase, such as for example T4 polynucleotideligase, or by another suitable chemical reaction. In a preferredembodiment, the 5′ end 58 of the second linker 50 additionally comprisesa 5′ pyrophosphate adenosine.

In one embodiment, the 5′ end 54 of the first linker 48, or the 3′ end56 of the second linker 50, or both the 5′ end 54 of the first linker 48and the 3′ end 56 of the second linker 50 comprise a label, such as forexample a fluorescent dye, to facilitate detection, as will beunderstood by those with skill in the art with reference to thisdisclosure. Further, the first linker 48, or the second linker 50, orboth the first linker 48 and the second linker 50 can comprise a label,such as for example a fluorescent dye, to facilitate detection at aposition other than at the 5′ end 54 of the first linker 48, or the 3′end 56 of the second linker 50, as long as the presence of the labeldoes not interfere with other steps of the present method, as will beunderstood by those with skill in the art with reference to thisdisclosure.

In one embodiment, the 5′ end 54 of first linker 48 comprises one ormore than one residue that extends beyond the 3′ end 18 of the firstadapter segment 12 after the first linker 48 hybridizes to the firstadapter segment 12. In one embodiment, the one or more than one residueof the 5′ end 54 of first linker 48 that extends beyond the 3′ end 18 ofthe first adapter segment 12 functions as a primer binding site thatallows the first linker 48 to be distinguished from the second linker 50in downstream amplification reactions. For example, in one embodiment,the first linker 48 comprises, from the 5′ end 54 to the 3′ end 52 offirst linker 48, a T3 promoter sequence, a short spacer sequence and aT7 promoter sequence, while the nucleotide residues of the first adaptersegment 12 consist of the substantial complement of the T7 promotersequence only.

In one embodiment, the 3′ end 56 of second linker 50 comprises one ormore than one residue that extends beyond the 5′ end 20 of the secondadapter segment 14 after the second linker 50 hybridizes to the secondadapter segment 14. In one embodiment, the one or more than one residueof the 3′ end 56 of second linker 50 that extends beyond the 5′ end 32of the second adapter segment 14 functions as a primer binding site thatallows the second linker 50 to be distinguished from the first linker 48in downstream amplification reactions. For example, in one embodiment,the second linker 50 comprises, from the 3′ end 56 to the 5′ end 58 ofsecond linker 50, a T3 promoter sequence, a short spacer sequence and anSP6 promoter sequence, while the nucleotide residues of the secondadapter segment 14 consist of the substantial complement of the SP6promoter sequence only.

After the first linker sequence of the first linker 48, and the secondlinker sequence of the second linker 50 are designed, the first linker48 and the second linker 50 can be synthesized according to standardtechniques, as will be understood by those with skill in the art withreference to this disclosure. Alternately, the first linker 48 and thesecond linker 50 can be purchased from a vendor of polynucleotide orpolynucleotide analog sequences, such as for example, from IntegratedDNA Technologies or Invitrogen Corp.

Referring now to FIG. 2, the method then comprises combining the captureprobe 10, the first linker segment 48, the second linker segment 50 andthe sample, represented in FIG. 2 by the miRNA of interest 42. In apreferred embodiment, the method comprises combining the sample, thecapture probe 10, the first linker 48 and the second linker 50 in asolution. The capture probe 10, the first linker 48, the second linker50 and the sample can be combined simultaneously, or sequentially in anyorder, as will be understood by those with skill in the art withreference to this disclosure. For example, the capture probe 10 iscombined with the sample first, and then the capture probe 10 and sampleare combined with the first linker 48 and second linker 50; oralternately for example, the capture probe 10, first linker 48 andsecond linker 50 are combined first, and then the capture probe 10,first linker 48 and second linker 50 are combined with the sample; oralternately for example, the first linker 48 and the second linker 50are combined with the sample first, and then the capture probe 10 iscombined with the first linker 48, second linker 50 and the sample.

In one embodiment, combining the capture probe 10, the first linker 48,the second linker 50 and the sample comprises combining approximatelyequimolar amounts of the capture probe 10, the first linker 48 and thesecond linker 50. In another embodiment, combining the capture probe 10,the first linker 48, the second linker 50 and the sample comprisescombining approximately equimolar amounts of the capture probe 10, thefirst linker 48 and the second linker 50 with an amount of sampleexpected to contain approximately one tenth the molar amount of miRNA ofinterest 42 as of the capture probe 10, the first linker 48 and thesecond linker 50. In another embodiment, combining the capture probe 10,the first linker 48, the second linker 50 and the sample comprisescombining approximately equimolar amounts of the capture probe 10, thefirst linker 48 and the second linker 50 with an amount of sampleexpected to contain approximately one half and one tenths and the molaramount of miRNA of interest 42 as of the capture probe 10, the firstlinker 48 and the second linker 50. In one embodiment, combining thecapture probe 10, the first linker 48, the second linker 50 and thesample comprises combining the sample with between 0.1 pmoles and 100pmoles/μl each of the capture probe 10, the first linker 48 and thesecond linker 50 in a suitable buffer to create a solution comprisingthe capture probe 10, the first linker 48, the second linker 50 and thesample. In a preferred embodiment, the buffer is selected from the groupconsisting of 1×TE buffer in 0.1-1 M sodium chloride, and 0.1 M MOPS in1 mM EDTA and 100 mM sodium chloride. As will be understood by thosewith skill in the art with reference to this disclosure, the pH selectedfor the buffer will be one that optimizes the intended reactions. Ingeneral, the pH selected will be between 6 and 8, preferably between 6.4and 7.4 and more preferably, near 7.0. In a preferred embodiment, themethod further comprises adding one or more than one. RNAse inhibitor tothe combination of the sample, the capture probe 10, the first linker48, the second linker 50, such as for example an RNAase inhibitorselected from the group consisting of lithium dodecylsulfate (LiDS), theammonium salt of tricarboxylic acid and sodium salt of aurinetricarboxylic acid.

Referring now to FIG. 3, after combining the capture probe 10, the firstlinker 48, the second linker 50 and the sample, the method comprisesallowing the first linker 48 to hybridize with the first adapter segment12, the miRNA of interest 42 to hybridize with the miRNA binding segment16, and the second linker 50 to hybridize with the second adaptersegment 14, thereby binding the first linker 48, the miRNA of interest42, and the second linker 50 to the capture probe 10. In one embodiment,allowing the first linker 48 to hybridize with the first adapter segment12, the miRNA of interest 42 to hybridize with the miRNA binding segment16, and the second linker 50 to hybridize with the second adaptersegment 14 comprises incubating the solution comprising first linker 48,the second linker 50, the capture probe 10 and the sample for between 1minute and 60 minutes at between 25° C. and 50° C. until substantiallyall of the miRNA of interest 42 has hybridized to the capture probes 10.

In one embodiment, the first adapter segment 12 comprises a solid phasebinding group 28, or the second adapter segment 14 comprises a solidphase binding group 40, or both the first adapter segment 12 comprises asolid phase binding group 28 and the second adapter segment 14 comprisesa solid phase binding group 40, as disclosed in this disclosure, and themethod further comprises binding the capture probe 10 to a solid phase(not shown) before or after combining the capture probe 10, the firstlinker 48, the second linker 50 and the sample. In one embodiment, thesolid phase is a plurality of beads where each bead has a diameter ofbetween 0.01μ and 5μ, though the solid phase can be any suitable solidphase as will be understood by those with skill in the art withreference to this disclosure. For example, in one embodiment, the solidphase binding group comprises biotin, and the solid phase isparamagnetic particles having a diameter of 1.0μ and comprisingstreptavidin immobilized to the surface of the particles.

In one embodiment, the capture probes 10 are bound to a solid phasethrough the first adapter segment 12 or through the second adaptersegment 14 or through both the first adapter segment 12 and the secondadapter segment 14, and the method further comprises hybridizing themiRNA of interest 42 to the miRNA binding segment 16 of the captureprobes 10 bound to the solid phase, and then incubating the captureprobes/miRNA of interest-bound to the solid phase with the first linker48 and second linker 50 under conditions sufficient to hybridize thefirst linker 48 to the first adapter segment 12 of the capture probe 10and the second linker 50 to the second adapter segment 14 of the captureprobe 10.

In a preferred embodiment, the first linker 48 hybridizes to the firstadapter segment 12 at a position where the last residue on the 3′ end 52of the first linker 48 hybridizes to a residue on the first adaptersegment 12 that is between 1 residue and 5 residues from the 3′ end 22of the miRNA binding segment 16. In a particularly preferred embodiment,the first linker 48 hybridizes to the first adapter segment 12 at aposition where the last residue on the 3′ end 52 of the first linker 48hybridizes to a residue on the first adapter segment 12 that isimmediately adjacent to the 3′ end 22 of the miRNA binding segment 16.

In a preferred embodiment, the second linker 50 hybridizes to the secondadapter segment 14 at a position where the last residue on the 5′ end 58of the second linker 50 hybridizes to a residue on the second adaptersegment 14 that is between 1 residue and 5 residues from the 5′ end 34of the miRNA binding segment 16. In a particularly preferred embodiment,the second linker 50 hybridizes to the second adapter segment 14 at aposition where the last residue on the 5′ end 58 of the second linker 50hybridizes to a residue on the second adapter segment 14 that isimmediately adjacent to the 5′ end 34 of the miRNA binding segment 16.

In one embodiment, the capture probe is bound to a solid phase throughthe first adapter segment 12 of through the second adapter segment 14 orthrough both the first adapter segment 12 and the second adapter segment14, and the method further comprises purifying the capture probe 10 withhybridized first linker 48, miRNA of interest 42 and second linker50-bound to the solid phase by removing non-hybridized first linkers 48,second linkers 50 and any other substances such as messenger RNAs,transfer RNAs, ribosomal RNAs and genomic DNA that are not bound to thesolid phase. In one embodiment, purifying comprises washing the captureprobe 10 with hybridized first linker 48, miRNA of interest 42 andsecond linker 50-bound to the solid phase with a suitable buffer, suchas for example 1×TE buffer in 0.1-1 M sodium chloride (pH 6.4-7.4,preferably pH 6.8-7.2). In another embodiment, the solid phase comprisesparamagnetic particles, the solid phase is contained in a vesselcomprising a surface and a cap, and purifying comprises applying amagnetic field to attract the solid phase to the surface of the vesselor the cap of the vessel. In a preferred embodiment, after applying themagnetic field to attract the solid phase to the surface of the vesselor the cap of the vessel, the method comprises washing the capture probe10 with hybridized first linker 48, miRNA of interest 42 and secondlinker 50-bound to the solid phase with a suitable buffer, such as forexample 0.1×TE buffer (pH 7.4).

Next, as show in FIG. 4, the method comprises covalently ligating the 3′end 52 of the first linker 48 that is hybridized to the first adaptersegment 12 to the 5′ end 46 of the miRNA of interest 42 that ishybridized to the miRNA binding segment 16, and then covalently ligatingthe 3′ end 44 of the miRNA of interest 42 that is hybridized to themiRNA binding segment 16 to the 5′ end 58 of the second linker 50 thatis hybridized to the second adapter segment 14. Ligation of the 3′ end52 of the first linker 48 to the 5′ end 46 of the miRNA of interest 42,and ligation of the 3′ end 44 of the miRNA of interest 42 to the 5′ end58 of the second linker 50 can be accomplished in any order, includingsimultaneously or sequentially. In one embodiment, the ligation isaccomplished by standard techniques, as will be understood by those withskill in the art with reference to this disclosure. In a preferredembodiment, the ligation comprises treating the capture probe 10 withthe hybridized first linker 48, miRNA of interest 42 and second linker50 with a suitable ligase, such as for example T4 polynucleotide ligasein the presence of suitable buffer and essential cofactors for asufficient time for the ligation to proceed to near total completion ofligation. As will be understood by those with skill in the art withreference to this disclosure, the presence of the first adapter segment12 and the second adapter segment 14 in the Capture probe 10 facilitatethe ligation of the first linker 48 and the second linker 50 to themiRNA of interest 42 by aligning the 3′ end 52 of the first linker 48with the 5′ end of the miRNA of interest 42, and aligning the 5′ end 58of the second linker 50 with the 3′ end 22 of the miRNA of interest 42.The ligation step produces a “complex 60” defined as a strand 62 offirst linker 48, miRNA of interest 42 and second linker 50 that havebeen ligated together (“ligated first linker 48-miRNA of interest42-second linker 50”), and where the strand 62 is hybridized to thecapture probe 10.

In one embodiment, the method further comprises purifying the complex60. In a preferred embodiment, purifying comprises washing the complex60 with a suitable buffer, such as for example T4 polynucleotide ligaseincubation buffer containing ATP (Promega Corp., Madison Wis. US)

In one embodiment, the complex 60 is bound to a solid phase through thefirst adapter segment 12 or through the second adapter segment 14 orthrough both the first adapter segment 12 and the second adapter segment14, and the method further comprises purifying the complex 60 byremoving non-hybridized first linkers 48, non-hybridized second linkers50 and any other substances such as messenger RNAs, transfer RNAs,ribosomal RNAs and genomic DNA that are not bound to the solid phase. Inone embodiment, purifying comprises washing the complex 60-bound to thesolid phase with a suitable buffer, such as for example 0.1×TE buffer(pH 7.4). In another embodiment, the solid phase comprises paramagneticparticles, and purifying comprises applying a magnetic field to attractthe complex 60-bound to the solid phase to the vessel containing thecomplex 60-bound to the solid phase. In a preferred embodiment, afterapplying the magnetic field, the method comprises washing the complex60-bound to the solid phase with a suitable buffer, such as for example0.1×TE buffer (pH 7.4).

Next, as shown in FIG. 5, the method comprises dehybridizing the strand62 of the ligated first linker 48-miRNA of interest 42-second linker 50from the capture probe 10. In one embodiment, dehybridization isaccomplished by standard techniques, as will be understood by those withskill in the art with reference to this disclosure. In a preferredembodiment, dehybridizing comprises applying a substance that abolishesor substantially reduces the hybridization between the capture probe 10and the strand 62 of the ligated first linker 48-miRNA of interest42-second linker 50. In a preferred embodiment, the complex is bound tothe solid phase, and dehybridizing comprises applying a low ionicstrength solution to the bound complex, such as for example a solutionof sterile nuclease free water warmed to 80° C., thereby producing asolution of the ligated first linker 48-miRNA of interest 42-secondlinker 50, and of capture probe 10-bound to the solid phase.

In one embodiment, the method further comprises purifying the ligatedfirst linker 48-miRNA of interest 42-second linker 50 that has beendehybridized from the capture probe 10. In a preferred embodiment,purifying the ligated first linker 48-miRNA of interest 42-second linker50 is accomplished according to standard techniques, as will beunderstood by those with skill in the art with reference to thisdisclosure. In a preferred embodiment, the capture probes 10 are boundto a solid phase, and purifying the ligated first linker 48-miRNA ofinterest 42-second linker 50 comprises separating the ligated firstlinker 48-miRNA of interest 42-second linker 50 from the capture probes10-bound to the solid phase by transferring a solution containing theligated first linker 48-miRNA of interest 42-second linker 50 afterdehybridization to a separate container. In another preferredembodiment, the first linker 48, or the second linker 50, or both thefirst linker 48 and the second linker 50 comprise nuclease resistantnucleotides, or comprise nucleotides with a phosphothioate backbone thatrender the first linker 48, or the second linker 50, or both the firstlinker 48 and the second linker 50 resistant to nuclease degradation,and purifying the ligated first linker 48-miRNA of interest 42-secondlinker 50 comprises applying DNAase to a solution containing the ligatedfirst linker 48-miRNA of interest 42-second linker 50 to destroy any DNApresent in the solution, thereby advantageously decreasing false signalsduring downstream amplification with PCR.

In another preferred embodiment, purifying the ligated first linker48-miRNA of interest 42-second linker 50 comprises circularizing theligated first linker 48-miRNA of interest 42-second linker 50.Circularizing the ligated first linker 48-miRNA of interest 42-secondlinker 50 can be accomplished by standard techniques, as will beunderstood by those with skill in the art with reference to thisdisclosure. In one embodiment, circularizing comprises treating theligated first linker 48-miRNA of interest 42-second linker 50 with aligase that catalyzes intramolecular ligation (i.e., circularization) ofsingle-stranded polynucleotide templates having a 5′-phosphate and a3′-hydroxyl group, such as for example CircLigase™ (EpicentreBiotechnologies, Madison, Wis. US). In another embodiment, circularizingcomprises treating the ligated first linker 48-miRNA of interest42-second linker 50 with a polynucleotide kinase, such as for example T4polynucleotide kinase, to phosphorylate the 5′ ends of the ligated firstlinker 48-miRNA of interest 42-second linker 50 before treating theligated first linker 48-miRNA of interest 42-second linker 50 with aligase that catalyzes intramolecular ligation. In one embodiment, themethod comprises circularizing the strand 62 of the ligated first linker48-miRNA of interest 42-second linker 50, and purifying the strand 62 ofthe ligated first linker 48-miRNA of interest 42-second linker 50comprises treating the solution containing the circularized strand 62 ofthe ligated first linker 48-miRNA of interest 42-second linker 50 withone or more than one exonuclease, such as for example exonuclease I fromE. coli to destroy any polynucleotides or polynueleotide analogs presentin the solution other than the circularized strand 62 of the ligatedfirst linker 48-miRNA of interest 42-second linker 50.

According to another embodiment of the present invention, there isprovided a method for identifying an miRNA of interest 42. In oneembodiment, the method for identifying an miRNA of interest 42comprises, first, isolating the miRNA of interest 42 according to thepresent invention. In one embodiment, the method further comprisessequencing the miRNA of interest 42 portion of the strand 62 of theligated first linker 48-miRNA of interest 42-second linker 50 bystandard techniques, as will be understood by those with skill in theart with reference to this disclosure. In a preferred embodiment, asshown in FIG. 6, sequencing the miRNA of interest 42 of the strand 62 ofthe ligated first linker 48-miRNA of interest 42-second linker 50comprises subjecting the strand 62 of the ligated first linker 48-miRNAof interest 42-second linker 50 to reverse transcription to produce adouble stranded product 64 comprising a first strand 62 of the ligatedfirst linker 48-miRNA of interest 42-second linker 50 and a secondstrand 66 that is the complement of the first strand 62, where thesecond strand is hybridized to the first strand. In one embodiment, thesecond strand 66 is a cDNA of the first strand. In one embodiment,subjecting the strand 62 of the ligated first linker 48-miRNA ofinterest 42-second linker 50 to reverse transcription to produce adouble stranded product 64 is accomplished by standard techniques, aswill be understood by those with skill in the art with reference to thisdisclosure. For example, in one embodiment, subjecting the strand 62 ofthe ligated first linker 48-miRNA of interest 42-second linker 50 toreverse transcription to produce a double stranded product 64 compriseshybridizing the second linker 50 to a substantially complementary primer68 having a primer sequence, and comprising between 16 and 25 residues,and further comprising a 3′ end 70 capable of being extended by theaction of a polynucleotide polymerase, such as a reverse transcriptase,that can use the strand 62 of the ligated first linker 48-miRNA ofinterest 42-second linker 50 as a template for extension and chainsynthesis. In one embodiment, part of the second linker sequence issubstantially complementary to a SP6 polynucleotide synthesis promotermotif, and the primer sequence comprises a SP6 promoter sequence thathybridizes to the part of the second linker segment sequence that issubstantially complementary to the SP6 polynucleotide synthesis promotermotif. In this embodiment, after the primer 68 has hybridized to thesecond linker 50, the method comprises contacting the strand 62 of theligated first linker 48-miRNA of interest 42-second linker 50 with thehybridized primer 68 with a reverse transcriptase, suitable buffers,cofactors and dNTPs (dA, dG, dC, dT or dU) to extend the primer 68,thereby producing the double stranded product 64. As will be understoodby those with skill in the art with reference to this disclosure, whenthe strand 62 of the ligated first linker 48-miRNA of interest 42-secondlinker 50 subjected to reverse transcription comprises bothribonucleotides and polynucleotides other than ribonucleotides, thepolymerase used to effect reverse transcription must be effective on allof the types of polynucleotides present in the strand 62 of the ligatedfirst linker 48-miRNA of interest 42-second linker 50.

In one embodiment, sequencing comprises amplifying the double strandedproduct 64 to produce amplification products. In a preferred embodiment,amplification is accomplished by standard techniques, as will beunderstood by those with skill in the art with reference to thisdisclosure. For example, in one embodiment, amplifying comprises usingPCR, according to techniques well known to those with skill in the art.

In one embodiment, the strand 62 of the ligated first linker 48-miRNA ofinterest 42-second linker 50 is circularized as disclosed above, thefirst linker sequence, or the second linker sequence, or both the firstlinker sequence and the second linker sequence comprise an N4 RNApolymerase promoter, and the method further amplifying the circularizedligated strand of the first linker 48-miRNA of interest 42-second linker50 with N4 RNA polymerase (Epicentre Biotechnologies) to produce RNArunoff sequences. The runoff sequences are linearly amplifiedrepresentations of the miRNAs in the sample such that the amounts ofmiRNAs relative to one another in the sample is the same as the amountsof runoff sequences produced relative to one another, though, of course,the concentration of the runoff sequences are increased with respect tothe concentrations of their corresponding miRNAs present in the sample.

In one embodiment, sequencing the miRNA of interest 42 of the strand ofligated first linker 48-miRNA of interest 42-second linker 50 comprisescloning the amplification products and culturing the amplificationproducts as isolated colonies to provide a library of miRNAs for furtherstudy or for the production of RNAi molecules for each miRNA, accordingto techniques well known to those with skill in the art, as will beunderstood by those with skill in the art with reference to thisdisclosure.

Example I Method for Isolating miRNAs

According to one embodiment of the present invention, the method forisolating microRNAs (miRNAs) was performed as follows. First, eightcapture probes were designed, SEQ ID NO:13 through SEQ ID NO:20 as shownin Table III, comprising first adapter segment sequences of SEQ ID NO:1where the 5′-most residue was biotinylated, second adapter segmentsequences of SEQ ID NO:2, and miRNA binding segment sequences that werethe exact complement to eight known human miRNAs from a public database.The capture probes were produced by Integrated DNA Technologiesaccording to embodiments of the present invention, and were resuspendedin 0.1×TE buffer with 2% Acetonitrile (Sigma Aldrich Corp.; St. Louis,Mo. US) for a final concentration of each capture probe of 100 pmol/μl.

TABLE III CAPTURE PROBE SEQUENCES miRNA Captured byCapture Probe Sequence the Capture SEQ ID NO: 5′-3′ Probe SEQ ID NO: 13ATTTAGGTGACACTATAGAAACTAT hsa-let-7a ACAACCTACTACCTCACCCTATAGTGAGTCGTATTA SEQ ID NO: 21 ATTTAGGTGACACTATAGAACTATA hsa-let-7eCAACCTCCTACCTCACCCTATAGTG AGTCGTATTA SEQ ID NO: 22ATTTAGGTGACACTATAGAGCTACC hsa-miR- TGCACTGTAAGCACTTTTCCCTATA 106aGTGAGTCGTATTA SEQ ID NO: 23 ATTTAGGTGACACTATAGACGCGTA hsa-miR-CCAAAAGTAATAATGCCCTATAGTG 126* AGTCGTATTA SEQ ID NO: 24ATTTAGGTGACACTATAGATCACAT hsa-miR- AGGAATAAAAAGCCATACCCTATAG 135aTGAGTCGTATTA SEQ ID NO: 25 ATTTAGGTGACACTATAGAGATTCA hsa-miR-138CAACACCAGCTCCCTATAGTGAGTC GTATTA SEQ ID NO: 26 ATTTAGGTGACACTATAGACGAAGGhsa-miR-154 CAACACGGATAACCTACCCTATAGT GAGTCGTATTA SEQ ID NO: 27ATTTAGGTGACACTATAGAAATAGG hsa-miR- TCAACCGTGTATGATTCCCTATAGT 154*GAGTCGTATTA

Next, eight miRNAs having sequences SEQ ID NO:28 through SEQ ID NO:35that were the exact complements of the miRNA binding segment sequencesSEQ ID NO:13, and SEQ ID NO: 21 through SEQ ID NO:27 of the captureprobes were obtained from Integrated DNA Technologies as indicated inTable IV. Each of the miRNAs as given in Table IV were prepared fromribonucleotides.

TABLE IV miRNAs ISOLATED BY THE METHOD miRNA Sequence 5′-3′ miRNA NameSEQ ID NO: 28 UGAGGUAGUAGGUUGUAUAGUU hsa-let-7a SEQ ID NO: 29UGAGGUAGGAGGUUGUAUAGU hsa-let-7e SEQ ID NO: 30 AAAAGUGCUUACAGUGCAGGUhsa-mi-106a AGC SEQ ID NO: 31 CAUUAUUACUUUUGGUACGCG hsa-mi-126*SEQ ID NO: 32 UAUGGCUUUUUAUUCCUAUGUGA hsa-mi-135a SEQ ID NO: 33UAGGUUAUCCGUGUUGCCUUCG hsa-mi-138 SEQ ID NO: 34 UAGGUUAUCCGUGUUGCCUUCGhsa-mi-154 SEQ ID NO: 35 AAUCAUACACGGUUGACCUAUU hsa-miR-154*

Then, each miRNA, SEQ ID NO:28 through SEQ ID NO was resuspended in astabilization buffer containing 1 mM Sodium Citrate pH 6.8 (Ambion,Inc.) and 1 mM aurine tricarboxylic acid (Sigma Aldrich Corp.) to afinal concentration of 100 pmol/μl for each miRNA. Next, each of themiRNAs, SEQ ID NO:28 through SEQ ID NO:35, was aliquoted into 10 μlworking stocks in 0.5 metal laminate 30 tubes (Nalge Nunc International;Rochester, N.Y. US) to reduce freeze-thaw effects.

Then, the miRNA (hsa-miR-138), SEQ ID NO:33, was hybridized to thecapture probe SEQ ID NO:25 by placing the following components into 2.0metal laminate 30 polypropylene screw cap tubes (Starstedt, Inc.;Newton, N.C. US): 10 pmol of the capture probe SEQ ID NO:25, 1 pmol ofmiRNA SEQ ID NO:33, 1 metal laminate 30 of 1× Lysis Buffer (5 mM aurinetricarboxcylic acid, 10 mM MOPS, 500 mM lithium chloride, and 10 mM EDTAand 1% SDS). Next, the tube was briefly pulsed in a centrifuge to mixthe components. Then, the tube was left at room temperature for 10minutes with occasional inversion of the tube to further mix thecomponents, at which time hybridization of the miRNA SEQ ID NO:33 to thecapture probe, SEQ ID NO:25, was essentially total, producing a “captureprobe with hybridized miRNA.”

Next, the capture probe with hybridized miRNA was coupled to a solidphase As indicated above, the capture probe portion, SEQ ID NO:25, ofthe capture probe with hybridized miRNA was biotinylated at the 5′ end.The capture probe with hybridized miRNA was coupled to a solid phase ofstreptavidin-coated, paramagnetic beads by adding 20 μl of StreptavidinMagneSphere® paramagnetic particles (Promega Corp.) to the 2.0 metallaminate 30 tube containing the capture probe with hybridized miRNA.Then, the tube was placed on a small tube rotator (Glas-Col, L.L.C.;Terre Haute, Ind. US) set at 20% for 30 minutes at room temperature,resulting in “capture probe with hybridized miRNA bound to the solidphase.”

Next, the capture probe with hybridized miRNA bound to the solid phasewas separated from the remaining components in the tube by adding amagnet assembly to the cap of the tube, and inverting the tube with themagnet assembly in order to collect the capture probe with hybridizedmiRNA bound to the solid phase in the cap. The tube with magnet assemblywas placed upright to permit fluid to drain from the cap, and then, themagnetic cap assembly was placed on a new 2.0 metal laminate 30 tubecontaining 200 μl of Wash Buffer A (10 mM Tris-HCl pH 7.5, 500 mM LiCl,10 mM EDTA pH 8, and 0.1% LiDS). Next, the capture probe with hybridizedmiRNA bound to the solid phase was resuspended in the Wash Buffer A byremoving the magnet from the cap and gently flicking the tube. Onceresuspended, the entire volume was transferred to a 0.45 micron Lidafilter spin column (Nalge Nunc International) that was placed in a 1.5metal laminate 30 collection tube. Then, the filter spin column in thecollection tube containing the capture probe with hybridized miRNA inWash Buffer A was centrifuged at 1,000×g for 1 minute, the flow throughdiscarded, and the filter spin column placed back in the collectiontube. Next, the capture probe with hybridized miRNA was washed by adding100 μl of the Wash Buffer B (10 mM Tris-HCl pH 7.5, 500 mM LiCl, and 10mM EDTA) to the filter spin column in the collection tube, followed byspinning the filter spin column for 1 minute at 1,000×g. The flowthrough was discarded and the filter spin column containing the captureprobe with hybridized miRNA was placed back in the collection tube.

To facilitate downstream analysis, a first linker segment having a firstlinker segment sequence of (5′-3′) taatacgactcactataggg, SEQ ID NO:36,which comprised a T7 promoter sequence, and a second linker having asecond linker segment sequence of (5′-3′) tctatagtgtcacctaaat, SEQ IDNO:37, which comprised a SP6 promoter sequence which was phosphorylatedat the 5′ end, were hybridized, respectively, to the first adaptersegment and second adapter segment of the capture probe portion of thecapture probe with hybridized miRNA; and the 3′ end of the first linkerwas ligated to the 5′ end of the miRNA that was hybridized to thecapture probe, and the 5′ end of the second linker was ligated to the 3′end of the miRNA that was hybridized to the capture probe, producing a“complex” of a strand of first linker, miRNA and second linker that wereligated together (“strand of ligated first linker-miRNA-second linker”),where the strand is hybridized to the capture probe. Hybridization andligation of the first linker segment and second linker segment wasperformed by adding 20 μl of ligation reaction mix 1× rapid ligasebuffer (Promega Corp.), 5 pmol of first linker segment, SEQ ID NO:36,(Integrated DNA Technologies), 5 pmol of second linker segment, SEQ IDNO:37, (Integrated DNA Technologies), and 1 unit of T4 polynucleotideligase (Promega Corp.) and 25% glycerol) to the filter spin column inthe collection tub, and allowing the mixture to stand at roomtemperature for 15 minutes. Then, the reaction was stopped by adding 200μl of Wash Buffer B and spinning at 1,000×g for 1 minute. The flowthrough was discarded, and the filter spin column was placed back intothe collection tube.

Next, in order to digest and remove excess first linker segments, SEQ IDNO:36, and second linker segments, SEQ ID NO:37, 20 μl of ExoSAP-IT™(USB Corp.; Cleveland, Ohio US) digest mix containing 17 μl of sterileDI H₂O, 1 μl of ExoSAP-IT™ and 2 μl of 10×PCR Buffer (AppliedBiosciences; Foster City, Calif. US) was added to each tube and placedin a 37° C. incubator for 30 minutes. The digested product was washedoff and removed by adding 200 μl of Wash Buffer B to each tube, andcentrifuging the tube at 1,000×g for 1 minute. The flow through wasdiscarded, and the filter spin column was placed back into thecollection tube.

Then, the capture probe, SEQ ID NO:25, was dehybridized from the strandof ligated first linker-miRNA-second linker by adding 20 μl of elutionbuffer (10 mM Tris-HCl, pH 7.5) that had been pre-heated to 80° C. tothe filter spin column, and incubating the filter spin column for 1minute at room temperature. Next, the filter spin column was spun at1,000×g for 1 minute. The flow through containing the strand of ligatedfirst linker-miRNA-second linker was removed from the collection tubeand placed into a new 1.5 metal laminate 30 screw cap tube (Starstedt,Inc.), and then stored in a −80° C. freezer until further use,completing the method for isolating miRNAs.

Example II Method for Identifying miRNAs

According to one embodiment of the present invention, the method foridentifying microRNAs (miRNAs) was performed as follows. First, an miRNAhaving an miRNA sequence of SEQ ID NO:33 was isolated as part of astrand of the ligated first linker-miRNA-second linker, as disclosed inExample I.

Next, a cDNA copy of the ligated first linker-miRNA-second linker wasmade by reverse transcription. Reverse transcription was performed by,first, annealing an SP6 primer, (5′-3′) CGATTTAGGTGACACTATAG, SEQ IDNO:38 (Integrated DNA Technologies) to the ligated firstlinker-miRNA-second linker, by adding 5 μl of the ligated firstlinker-miRNA-second linker to 1 μl of 100 pmol of the SP6 primer, 1 μlof dNTP mix containing 100 mM of each dNTP (Promega Corp.), and 7 μl ofsterile DI H₂O. These components were put into a 0.7 metal laminate 30PCR reaction tube (Applied Biosystems) and placed on an MJ ResearchThermocycler (Bio-Rad Inc.; Hercules, Calif. US) using calculatedcontrol and heated lid at 65° C. for 5 minutes, and then immediatelyplacing the tube on ice for 1 minute. While on ice, Superscript™ III(Invitrogen Corp.) reaction mix (4 μl 5× First Strand Buffer, 1 μl 0.1 MDTT, and 1 μl of 200 units/μl Superscript™ III Reverse Transcriptase)was added to the tube, and then briefly pulsed in a centrifuge to mixthe components. Then, cDNA synthesis was performed by incubating thetube on a thermocycler at 50° C. for 30 minutes, and heating to 70° C.for 15 minutes to terminate the reaction.

Next, the cDNA that was synthesized was amplified by PCR. PCR wasperformed using 5 μl of the cDNA and a PCR buffer containing 10 pmoleseach of a forward primer (T7), (5′-3′) TAATACGACTCACTATAGGG, SEQ IDNO:39, and a reverse primer (SP6), (5′-3′) CGATTTAGGTGACACTATAG, SEQ IDNO:38, 10% 10×PCR buffer (PE Biosystems; Foster City, Calif. US), 2 mMMgCl₂ (PE Biosystems), 2% Dimethyl Sulfoxide (Sigma Aldrich Corp.), 5 mMDTT (Bio-Rad Inc.; Hercules, Calif. US), 200 uM of each dNTP (PromegaCorp.), and 0.625 units of TaqGold (PE Biosystems) in a total volume of20 μl. These reaction components were assembled in a 96-well multiplate(Bio-Rad Inc.), and briefly pulsed in a centrifuge to mix components andplaced on a thermocycler (Bio-Rad Inc.). Cycling was performed usingcalculated control and a heated lid with cycles comprising 95° C. for 12minutes, followed by 30 cycles comprising 95° C. for 30 seconds, 53.5°C. for 20 seconds, 72° C. for 30 seconds, with a final extension at 72°C. for 6 minutes.

Then, electrophoresis was performed to determine the quality of theamplicons using 2 μl of PCR product run on precast Nuseive/GTG 3:1agarose gels containing ethidium bromide (BMA Corp.; Rockland, Me. US).The band observed on the gel was consistent with size and intensity forthe expected amplification polynucleotide product.

Next, in order to digest excess primers, 5 μl of ExoSAP-IT™ (USB Corp.)digest mix (3.25 μl of sterile DI H₂O, 1.5 μl of ExoSAP-IT™ and 0.25 μlof 100× Acetylated Bovine Serum Albumin (Promega Corp.) per 20 μlreaction) was then added to each well. The plates were then brieflypulsed in a centrifuge to mix components, sealed, and placed on thethermocycler. Incubation was performed using block control and a heatedlid with cycles comprising 37° C. for one hour, 65° C. for 10 minutes,and 80° C. for 10 minutes.

Then, the amplified cDNA was cloned into a vector and allowed to grow.Cloning of the amplified cDNA was performed using Zero Blunt® TOPO® PCRCloning Kit (Invitrogen Corp.). 0.5 μl of PCR product was added to 0.8μl pCR®-Blunt II-TOPO® vector in a 1.5 metal laminate 30 tube(Starstedt, Inc.), briefly pulsed in a centrifuge to mix components, andincubated at room temperature for 5 minutes. Next, 50 μl of competentDH5alpha-T1 cells was added to the tube and placed on a thermocyclerusing calculated control and heated lid, and a program comprising 4° C.for 30 minutes, 42° C. for 30 seconds, 15° C. for 4 minutes and 4° C.for 10 minutes. After the transformation was complete, 750 μl ofCircleGrow® (Qbiogene, Carlsbad, Calif. US) media with 100 ug/metallaminate 30 Ampicillin (Sigma Aldrich Corp.) was added to thetransformation reaction. Next, the tubes were placed in a shaker at 200×rpm at 37° C. for 90 minutes.

Then, the cloned cDNA was amplified by PCR by, first, lysing 1 μl of thetransformed DH5alpha culture with 4 μl of Sterile DI H2O in 0.2 metallaminate 30 PCR strip tubes (Fisher Scientific International, Inc.;Hampton, N.H. US) using the thermocycler programmed to 80° C. for 5minutes, 95° C. for 5 minutes and 4° C. for 5 minutes. Amplification wasconducted using 5 μl of the lysed clone culture and a PCR buffercontaining 10 pmoles each of forward primer M13-20, (5′-3′)GTAAAACGACGGCCAGTG, SEQ ID NO:40, and the reverse primer M13 REV.(5′-3′) GGAAACAGCTATGACCATGA, SEQ ID NO:41, 10% 10×PCR buffer (PEBiosystems), 2 mM MgCl₂ (PE Biosystems), 2% Dimethyl Sulfoxide (SigmaAldrich Corp.), 5 mM DTT (Bio-Rad Inc.), 200 uM of each dNTP (PromegaCorp.), and 0.625 units of TaqGold (PE Biosystems) in a total volume of20 μl. The reaction components were assembled in 96-well multiplate(Bio-Rad Inc.) and briefly pulsed in a centrifuge to mix the components.Cycling was performed using calculated control and a heated lid withcycles comprising 95° C. for 12 minutes, followed by 35 cyclescomprising 95° C. for 30 seconds, 59.2° C. for 20 seconds, 72° C. for 30seconds, with a final extension at 72° C. for 6 minutes.

Then, electrophoresis was performed to determine the quality of theamplicons using 2 μl of PCR product run on precast Nuseive/GTG 3:1agarose gels containing ethidium bromide (BMA Corp.). The band observedon the gel was consistent with size and intensity for the expectedamplifcation polynucleotide product.

Next, in order to digest excess primers, 5 1 of ExoSAP-IT™ (USB Corp.)digest mix (3.25 1 of sterile DI H2O, 1.5 1 of ExoSAP-IT™ and 0.25 1 of100× Acetylated Bovine Serum Albumin (Promega Corp.) per 20 1 reaction)was then added to each well. The plates were then briefly pulsed in acentrifuge to mix components, sealed, and placed on the thermocycler.Incubation was performed using block control and a heated lid withcycles comprising 37° C. for one hour, 65° C. for 10 minutes, and 80° C.for 10 minutes.

Then, the amplified cDNA was sequenced using 3 μl of each amplicon, 1.4pmoles each of primer SEQ ID NO:40 and SEQ ID NO:41, and 2 μl of BigDyeTerminator Ready Reactions mix version 3.0® (Applied Biosystems) per 10μl reaction. The reactions were set up using each PCR primer in both theforward and reverse orientation. The reaction components were assembledin MJ Research 96-well Multiplate and briefly pulsed in a centrifuge tomix. Cycling was performed using calculated control and a heated lidwith cycles comprising 95° C. for 5 minutes, followed by 35 cyclescomprising 95° C. for 30 seconds, 55° C. for 20 seconds, and 60° C. for4 minutes.

The finished sequence reaction plate was pulsed in a centrifuge and 1unit of shrimp alkaline phosphatase (USB Corp.) was added to each well.The plate was pulsed again and incubated at 37° C. for 30 minutes. Next,10 μl of 10% 1-Butanol was added to each well. The plate was then pulsedto mix and samples were transferred to a Sephadex® (Sigma ChemicalCorp.) matrix for dye removal. The Sephadex® matrix was constructed byfilling the wells of a 45 μl Multiscreen Column Loader® (MilliporeCorp.; Billerica, Mass. US) inverting it into a Multiscreen Plate®(Millipore) and filling each well with 300 μl DI H₂0 followed byplacement at 4° C. for a minimum of 24 hours prior to use to allow thegel to completely swell. Before use, excess water was spun out of theplate by centrifugation at 900×g for 5 minutes using the S2096 rotor onan Allegra™ 21 Centrifuge (Beckman Coulter Inc.; Fullerton, Calif. US)After samples were transferred to the Sephadex® matrix, a MicroAmpOptical 96-well Reaction Plate (Applied Biosystems) was placed under theSephadex® plate and the cleaned samples were collected by spinning thetwo plates again at 900×g for 5 minutes. The plate containing thecollected samples, was spun in a SpeedVac® (Telechem International,Inc.) until completely dried. 10 μl of DI Formamide was added to eachwell and the plate was cycled on a thermalcycler at 95° C. for 5minutes, 80° C. for 5 minutes, and 4° C. for 5 minutes to resuspend anddenature the cDNA. Then, the plate was placed on an ABI Prism® 3700 DNAAnalyzer (Applied Biosystems) using Dye Set “H,” mobility file“DT3700Pop5(BDv3)v1.mob,” cuvette temperature 48° C., injection time2000 seconds, and injection temperature 45° C. Sequences were thenanalyzed using Sequencher 4.5 (Gene Codes Corp., Ann Arbor, Mich. US)for basecalling and contig alignment.

Referring now to FIG. 7, there is shown a sequence trace of the cDNA,indicating that the miRNA (hsa-miR-138), SEQ ID NO:33 was successfullyisolated and identified by the methods of the present invention.

Using techniques corresponding to the above examples, all of the eightcapture probes of the capture probes SEQ ID NO:13, and SEQ ID NO:21through SEQ ID NO:27 were evaluated in a variety of differentcombinations with one another with respect to their ability to isolatetheir corresponding synthesized miRNAs, SEQ ID NO:28 through SEQ IDNO:35, and the capture probes were found to be both selective andspecific for the isolation of their corresponding synthetic miRNAsaccording to the present invention.

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

1-86. (canceled)
 87. A method for isolating a microRNA of interest froma sample comprising the microRNA of interest; the method comprising: a)providing a sample comprising the microRNA of interest; b) providing acapture probe comprising: i) a first adapter segment having a firstadapter segment sequence, the first adapter segment comprising a 3′ endand a 5′ end; ii) a second adapter segment having a second adaptersegment sequence, the second adapter segment comprising a 3′ end and a5′ end; and iii) a microRNA binding segment having a microRNA bindingsegment sequence; where the microRNA binding segment is substantiallycomplementary to, and capable of hybridizing to, one or more than onemicroRNA of interest by Watson-Crick base pairing; where the 5′ end ofthe first adapter segment is connected to the 3′ end of the microRNAbinding segment; and where the 3′ end of the second adapter segment isconnected to the 5′ end of the microRNA binding segment; c) providing afirst linker, wherein the first linker has a first linker sequence,comprises a 3′ end and a 5′ end, and is substantially complementary to,and capable of hybridizing to, the first adapter segment of the captureprobe; and wherein the 5′ end of first linker comprises one or more thanone residue that extends beyond the 3′ end of the first adapter segmentafter the first linker hybridizes to the first adapter segment; d)providing a second linker, wherein the second linker has a second linkersequence, comprises a 3′ end and a 5′ end, and is substantiallycomplementary to, and capable of hybridizing to, the second adaptersegment of the capture probe; e) combining the sample, the captureprobe, the first linker and the second linker in a solution; f) allowingthe first linker to hybridize with the first adapter segment, themicroRNA of interest to hybridize with the microRNA binding segment, andthe second linker to hybridize with the second adapter segment; g)ligating the 3′ end of the first linker to the 5′ end of the microRNA ofinterest, and ligating the 3′ end of the microRNA of interest to the 5′end of the second linker, thereby producing a complex defined as astrand of first linker, microRNA of interest and second linker that havebeen ligated together (ligated first linker-microRNA of interest-secondlinker) and that is hybridized to the capture probe; h) dehybridizingthe capture probe from the strand of the ligated first linker-microRNAof interest-second linker; and i) purifying the ligated firstlinker-microRNA of interest-second linker, thereby purifying themicroRNA of interest.
 88. The method of claim 87, where the sampleprovided comprises a plurality of microRNAs of interest; and where eachof the plurality of microRNAs of interest has microRNA of interestsequences that are identical to one another.
 89. The method of claim 87,where the sample provided comprises a plurality of microRNAs ofinterest; and where each of the plurality of microRNAs of interest hasmicroRNA of interest sequences that are different from one another. 90.The method of claim 87, where the first linker segment and the secondlinker segment comprise a substance selected from the group consistingof one or more than one type of polynucleotide, one or more than onetype of polynucleotide analog, and a combination of one or more than onetype of polynucleotide and polynucleotide analog.
 91. The method ofclaim 87, where the first linker, or the second linker, or both thefirst linker and the second linker are resistant to nucleasedegradation.
 92. The method of claim 87, where the first linker, or thesecond linker, or both the first linker and the second linker comprisenucleotides with a phosphothioate backbone that render the first linker,or the second linker, or both the first linker and the second linkerresistant to nuclease degradation.
 93. The method of claim 87, where thefirst linker, or the second linker, or both the first linker and thesecond linker comprise nuclease resistant nucleotides and comprisenucleotides with a phosphothioate backbone that render the first linker,or the second linker, or both the first linker and the second linkerresistant to nuclease degradation.
 94. The method of claim 87, where the5′ end of the first linker, or the 3′ end of the second linker, or boththe 5′ end of the first linker and the 3′ end of the second linkercomprise a label.
 95. The method of claim 87, where the first adaptersegment comprises a solid phase binding group, or the second adaptersegment comprises a solid phase binding group, or both the first adaptersegment comprises a solid phase binding group and the second adaptersegment comprises a solid phase binding group; and where the methodfurther comprises binding the capture probe to a solid phase before orafter combining the sample, the capture probe, the first linker and thesecond linker.
 96. The method of claim 95, where the solid phase is aplurality of paramagnetic particles.
 97. The method of claim 95, wherethe capture probe is bound to a solid phase through the first adaptersegment or through the second adapter segment or through both the firstadapter segment and the second adapter segment; and where the methodfurther comprises purifying the capture probes with hybridized firstlinker, microRNA of interest and second linker-bound to the solid phaseby removing non-hybridized first linkers, second linkers and any othersubstances that are not bound to the solid phase.
 98. The method ofclaim 87, where the first linker, or the second linker, or both thefirst linker and the second linker comprise nuclease resistantnucleotides, or comprise nucleotides with a phosphothioate backbone thatrender the first linker, or the second linker, or both the first linkerand the second linker resistant to nuclease degradation; and wherepurifying the ligated first linker-microRNA of interest-second linkercomprises applying DNAase to a solution containing the ligated firstlinker-microRNA of interest-second linker to destroy any DNA present inthe solution.
 99. The method of claim 87, where purifying the ligatedfirst linker-microRNA of interest-second linker comprises circularizingthe ligated first linker-microRNA of interest-second linker.
 100. Amethod for identifying a microRNA of interest, the method comprising: a)isolating the microRNAs according to claim 87; and b) sequencing themicroRNA of interest portion of the strand of the ligated firstlinker-microRNA of interest-second linker.
 101. A capture probe suitablefor use with a method for isolating miRNAs, the capture probecomprising: a) a first adapter segment having a first adapter segmentsequence, the first adapter segment comprising a 3′ end and a 5′ end; b)a second adapter segment having a second adapter segment sequence, thesecond adapter segment comprising a 3′ end and a 5′ end; and c) amicroRNA binding segment having a microRNA binding segment sequence;where the microRNA binding segment is substantially complementary to,and capable of hybridizing to, one or more than one microRNA of interestby Watson-Crick base pairing; where the 5′ end of the first adaptersegment is connected to the 3′ end of the microRNA binding segment; andwhere the 3′ end of the second adapter segment is connected to the 5′end of the microRNA binding segment.
 102. The capture probe of claim101, comprising a substance selected from the group consisting of one ormore than one type of polynucleotide, one or more than one type ofpolynucleotide analog, and a combination of one or more than one type ofpolynucleotide and polynucleotide analog.
 103. The capture probe ofclaim 101, where the first adapter segment, or the second adaptersegment, or both the first adapter segment and the second adaptersegment further comprise a sequence that is a polynucleotide synthesispromoter motif for a polynucleotide polymerase, or that is complementaryto a polynucleotide synthesis promoter motif for a polynucleotidepolymerase.
 104. The capture probe of claim 103, where thepolynucleotide synthesis promoter motif is a motif for a polynucleotidesynthesis promoter selected from the group consisting of T7, SP6, a T3DNA dependent RNA polymerase, a type 2 RNA polymerase of E. coli andsingle stranded DNA dependent N4 RNA polymerase.
 105. The capture probeof claim 101, where the first adapter segment, or the second adaptersegment, or both the first adapter segment and the second adaptersegment further comprise a restriction site motif.
 106. The captureprobe of claim 101, where the first adapter segment, or the secondadapter segment, or both the first adapter segment and the secondadapter segment further comprise a solid phase binding group toimmobilize the capture probe to a solid phase.